Nanoparticles having oligonucleotides attached thereto and uses therefor

ABSTRACT

The invention provides methods of detecting a nucleic acid. The methods comprise contacting the nucleic acid with one or more types of particles having oligonucleotides attached thereto. In one embodiment of the method, the oligonucleotides are attached to nanoparticles and have sequences complementary to portions of the sequence of the nucleic acid. A detectable change (preferably a color change) is brought about as a result of the hybridization of the oligonucleotides on the nanoparticles to the nucleic acid. The invention also provides compositions and kits comprising particles. The invention further provides methods of synthesizing unique nanoparticle-oligonucleotide conjugates, the conjugates produced by the methods, and methods of using the conjugates. In addition, the invention provides nanomaterials and nanostructures comprising nanoparticles and methods of nanofabrication utilizing nanoparticles. Finally, the invention provides a method of separating a selected nucleic acid from other nucleic acids.

[0001] This application is a continuation-in-part of pending applicationSer. No. 09/344,667, filed Jun. 25, 1999, which was acontinuation-in-part of pending application Ser. No. 09/240,755, filedJan. 29, 1999, which was a continuation-in-part of pending PCTapplication PCT/US97/12783, which was filed Jul. 21, 1997. Benefit ofprovisional applications No. 60/031,809, filed July 29, 1996, and No.60/200,161, filed Apr. 26, 2000 is also hereby claimed.

[0002] This invention was made with government support under NationalInstitutes Of Health (NIH) grant GM10265 and Army Research Office (ARO)grant DAAG55-0967-1-0133. The government has certain rights in theinvention.

FIELD OF THE INVENTION

[0003] The invention relates to methods of detecting nucleic acids,whether natural or synthetic, and whether modified or unmodified. Theinvention also relates to materials for detecting nucleic acids andmethods of making those materials. The invention further relates tomethods of nanofabrication. Finally, the invention relates to methods ofseparating a selected nucleic acid from other nucleic acids.

BACKGROUND OF THE INVENTION

[0004] The development of methods for detecting and sequencing nucleicacids is critical to the diagnosis of genetic, bacterial, and viraldiseases. See Mansfield, E. S. et al. Molecular and Cellular Probes, 9,145-156 (1995). At present, there are a variety of methods used fordetecting specific nucleic acid sequences. Id. However, these methodsare complicated, time-consuming and/or require the use of specializedand expensive equipment. A simple, fast method of detecting nucleicacids which does not require the use of such equipment would clearly bedesirable.

[0005] A variety of methods have been developed for assembling metal andsemiconductor colloids into nanomaterials. These methods have focused onthe use of covalent linker molecules that possess functionalities atopposing ends with chemical affinities for the colloids of interest. Oneof the most successful approaches to date, Brust et al., Adv. Mater., 7,795-797 (1995), involves the use of gold colloids and well-establishedthiol adsorption chemistry, Bain & Whitesides, Angew. Chem. Int. Ed.Engl., 28, 506-512(1989) and Dubois & Nuzzo, Annu. Rev. Phys. Chem., 43,437-464 (1992). In this approach, linear alkanedithiols are used as theparticle linker molecules. The thiol groups at each end of the linkermolecule covalently attach themselves to the colloidal particles to formaggregate structures. The drawbacks of this method are that the processis difficult to control and the assemblies are formed irreversibly.Methods for systematically controlling the assembly process are neededif the materials properties of these structures are to be exploitedfully.

[0006] The potential utility of DNA for the preparation of biomaterialsand in nanofabrication methods has been recognized. In this work,researchers have focused on using the sequence-specific molecularrecognition properties of oligonucleotides to design impressivestructures with well-defined geometric shapes and sizes. Shekhtman etal., New J. Chem., 17, 757-763 (1993); Shaw & Wang, Science, 260,533-536 (1993); Chen et al., J. Am Chem. Soc., 111, 6402-6407 (1989);Chen & Seeman, Nature, 350, 631-633 (1991); Smith and Feigon, Nature,356, 164-168 (1992); Wang et al., Biochem., 32, 1899-1904 (1993); Chenet al., Biochem., 33,13540-13546 (1994); Marsh et al., Nucleic AcidsRes., 23, 696-700 (1995); Mirkin, Annu. Review Biophys. Biomol. Struct.,23, 541-576 (1994); Wells, J. Biol. Chem., 263, 1095-1098 (1988); Wanget al., Biochem., 30, 5667-5674 (1991). However, the theory of producingDNA structures is well ahead of experimental confirmation. Seeman etal., New J. Chem., 17, 739-755 (1993).

SUMMARY OF THE INVENTION

[0007] The invention provides methods of detecting nucleic acids. In oneembodiment, the method comprises contacting a nucleic acid with a typeof nanoparticles having oligonucleotides attached thereto(nanoparticle-oligonucleotide conjugates). The nucleic acid has at leasttwo portions, and the oligonucleotides on each nanoparticle have asequence complementary to the sequences of at least two portions of thenucleic acid. The contacting takes place under conditions effective toallow hybridization of the oligonucleotides on the nanoparticles withthe nucleic acid. The hybridization of the oligonucleotides on thenanoparticles with the nucleic acid results in a detectable change.

[0008] In another embodiment, the method comprises contacting a nucleicacid with at least two types of nanoparticles having oligonucleotidesattached thereto. The oligonucleotides on the first type ofnanoparticles have a sequence complementary to a first portion of thesequence of the nucleic acid. The oligonucleotides on the second type ofnanoparticles have a sequence complementary to a second portion of thesequence of the nucleic acid. The contacting takes place underconditions effective to allow hybridization of the oligonucleotides onthe nanoparticles with the nucleic acid, and a detectable change broughtabout by this hybridization is observed.

[0009] In a further embodiment, the method comprises providing asubstrate having a first type of nanoparticles attached thereto. Thefirst type of nanoparticles has oligonucleotides attached thereto, andthe oligonucleotides have a sequence complementary to a first portion ofthe sequence of a nucleic acid. The substrate is contacted with thenucleic acid under conditions effective to allow hybridization of theoligonucleotides on the nanoparticles with the nucleic acid. Then, asecond type of nanoparticles having oligonucleotides attached thereto isprovided. The oligonucleotides have a sequence complementary to one ormore other portions of the sequence of the nucleic acid, and the nucleicacid bound to the substrate is contacted with the second type ofnanoparticle-oligonucleotide conjugates under conditions effective toallow hybridization of the oligonucleotides on the second type ofnanoparticles with the nucleic acid. A detectable change may beobservable at this point. The method may further comprise providing abinding oligonucleotide having a selected sequence having at least twoportions, the first portion being complementary to at least a portion ofthe sequence of the oligonucleotides on the second type ofnanoparticles. The binding oligonucleotide is contacted with the secondtype of nanoparticle-oligonucleotide conjugates bound to the substrateunder conditions effective to allow hybridization of the bindingoligonucleotide to the oligonucleotides on the nanoparticles. Then, athird type of nanoparticles having oligonucleotides attached thereto,the oligonucleotides having a sequence complementary to the sequence ofa second portion of the binding oligonucleotide, is contacted with thebinding oligonucleotide bound to the substrate under conditionseffective to allow hybridization of the binding oligonucleotide to theoligonucleotides on the nanoparticles. Finally, the detectable changeproduced by these hybridizations is observed.

[0010] In yet another embodiment, the method comprises contacting anucleic acid with a substrate having oligonucleotides attached thereto,the oligonucleotides having a sequence complementary to a first portionof the sequence of the nucleic acid. The contacting takes place underconditions effective to allow hybridization of the oligonucleotides onthe substrate with the nucleic acid. Then, the nucleic acid bound to thesubstrate is contacted with a first type of nanoparticles havingoligonucleotides attached thereto, the oligonucleotides having asequence complementary to a second portion of the sequence of thenucleic acid. The contacting takes place under conditions effective toallow hybridization of the oligonucleotides on the nanoparticles withthe nucleic acid. Next, the first type of nanoparticle-oligonucleotideconjugates bound to the substrate is contacted with a second type ofnanoparticles having oligonucleotides attached thereto, theoligonucleotides on the second type of nanoparticles having a sequencecomplementary to at least a portion of the sequence of theoligonucleotides on the first type of nanoparticles, the contactingtaking place under conditions effective to allow hybridization of theoligonucleotides on the first and second types of nanoparticles.Finally, a detectable change produced by these hybridizations isobserved.

[0011] In another embodiment, the method comprises contacting a nucleicacid with a substrate having oligonucleotides attached thereto, theoligonucleotides having a sequence complementary to a first portion ofthe sequence of the nucleic acid. The contacting takes place underconditions effective to allow hybridization of the oligonucleotides onthe substrate with the nucleic acid. Then, the nucleic acid bound to thesubstrate is contacted with liposomes having oligonucleotides attachedthereto, the oligonucleotides having a sequence complementary to aportion of the sequence of the nucleic acid. This contacting takes placeunder conditions effective to allow hybridization of theoligonucleotides on the liposomes with the nucleic acid. Next, theliposome-oligonucleotide conjugates bound to the substrate are contactedwith a first type of nanoparticles having at least a first type ofoligonucleotides attached thereto. The first type of oligonucleotideshave a hydrophobic group attached to the end not attached to thenanoparticles, and the contacting takes place under conditions effectiveto allow attachment of the oligonucleotides on the nanoparticles to theliposomes as a result of hydrophobic interactions. A detectable changemay be observable at this point. The method may further comprisecontacting the first type of nanoparticle-oligonucleotide conjugatesbound to the liposomes with a second type of nanoparticles havingoligonucleotides attached thereto. The first type of nanoparticles havea second type of oligonucleotides attached thereto which have a sequencecomplementary to at least a portion of the sequence of theoligonucleotides on the second type of nanoparticles, and theoligonucleotides on the second type of nanoparticles having a sequencecomplementary to at least a portion of the sequence of the second typeof oligonucleotides on the first type of nanoparticles. The contactingtakes place under conditions effective to allow hybridization of theoligonucleotides on the first and second types of nanoparticles. Then, adetectable change is observed.

[0012] In another embodiment, the method comprises contacting a nucleicacid to be detected with a substrate having oligonucleotides attachedthereto. The oligonucleotides have a sequence complementary to a firstportion of the sequence of said nucleic acid, the contacting takes placeunder conditions effective to allow hybridization of theoligonucleotides on the substrate with said nucleic acid. Next, saidnucleic acid bound to the substrate is contacted with a type ofnanoparticles having oligonucleotides attached thereto. Theoligonucleotides have a sequence complementary to a second portion ofthe sequence of said nucleic acid. The contacting takes place underconditions effective to allow hybridization of the oligonucleotides onthe nanoparticles with said nucleic acid. Then, the substrate iscontacted with silver stain to produce a detectable change, and thedetectable change is observed.

[0013] In yet another embodiment, the method comprises providing asubstrate having a first type of nanoparticles attached thereto. Thenanoparticles have oligonucleotides attached thereto, theoligonucleotides having a sequence complementary to a first portion ofthe sequence of a nucleic acid to be detected. Then, the nucleic acid iscontacted with the nanoparticles attached to the substrate underconditions effective to allow hybridization of the oligonucleotides onthe nanoparticles with said nucleic acid. Next, an aggregate probecomprising at least two types of nanoparticles having oligonucleotidesattached thereto is provided. The nanoparticles of the aggregate probeare bound to each other as a result of the hybridization of some of theoligonucleotides attached to them. At least one of the types ofnanoparticles of the aggregate probe have oligonucleotides attachedthereto which have a sequence complementary to a second portion of thesequence of said nucleic acid. Finally, said nucleic acid bound to thesubstrate is contacted with the aggregate probe under conditionseffective to allow hybridization of the oligonucleotides on theaggregate probe with said nucleic acid, and a detectable change isobserved.

[0014] In a further embodiment, the method comprises providing asubstrate having oligonucleotides attached thereto. The oligonucleotideshave a sequence complementary to a first portion of the sequence of anucleic acid to be detected. An aggregate probe comprising at least twotypes of nanoparticles having oligonucleotides attached thereto isprovided. The nanoparticles of the aggregate probe are bound to eachother as a result of the hybridization of some of the oligonucleotidesattached to them. At least one of the types of nanoparticles of theaggregate probe have oligonucleotides attached thereto which have asequence complementary to a second portion of the sequence of saidnucleic acid. The nucleic acid, the substrate and the aggregate probeare contacted under conditions effective to allow hybridization of saidnucleic acid with the oligonucleotides on the aggregate probe and withthe oligonucleotides on the substrate, and a detectable change isobserved.

[0015] In a further embodiment, the method comprises providing asubstrate having oligonucleotides attached thereto. An aggregate probecomprising at least two types of nanoparticles having oligonucleotidesattached thereto is provided. The nanoparticles of the aggregate probeare bound to each other as a result of the hybridization of some of theoligonucleotides attached to them. At least one of the types ofnanoparticles of the aggregate probe have oligonucleotides attachedthereto which have a sequence complementary to a first portion of thesequence of a nucleic acid to be detected. A type of nanoparticleshaving at least two types of oligonucleotides attached thereto isprovided The first type of oligonucleotides has a sequence complementaryto a second portion of the sequence of said nucleic acid, and the secondtype of oligonucleotides has a sequence complementary to at least aportion of the sequence of the oligonucleotides attached to thesubstrate. The nucleic acid, the aggregate probe, the nanoparticles andthe substrate are contacted under conditions effective to allowhybridization of said nucleic acid with the oligonucleotides on theaggregate probe and on the nanoparticles and hybridization of theoligonucleotides on the nanoparticles with the oligonucleotides on thesubstrate, and a detectable change is observed.

[0016] In another embodiment, the method comprises contacting a nucleicacid to be detected with a substrate having oligonucleotides attachedthereto. The oligonucleotides have a sequence complementary to a firstportion of the sequence of said nucleic acid. The contacting takes placeunder conditions effective to allow hybridization of theoligonucleotides on the substrate with said nucleic acid. The nucleicacid bound to the substrate is contacted with liposomes havingoligonucleotides attached thereto, the oligonucleotides having asequence complementary to a portion of the sequence of said nucleicacid. The contacting takes place under conditions effective to allowhybridization of the oligonucleotides on the liposomes with said nucleicacid. An aggregate probe comprising at least two types of nanoparticleshaving oligonucleotides attached thereto is provided. The nanoparticlesof the aggregate probe are bound to each other as a result of thehybridization of some of the oligonucleotides attached to them, at leastone of the types of nanoparticles of the aggregate probe havingoligonucleotides attached thereto which have a hydrophobic groupattached to the end not attached to the nanoparticles. The liposomesbound to the substrate are contacted with the aggregate probe underconditions effective to allow attachment of the oligonucleotides on theaggregate probe to the liposomes as a result of hydrophobicinteractions, and a detectable change is observed.

[0017] In yet another embodiment, the method comprises providing asubstrate having oligonucleotides attached thereto. The oligonucleotideshaving a sequence complementary to a first portion of the sequence of anucleic acid to be detected. A core probe comprising at least two typesof nanoparticles is provided. Each type of nanoparticles hasoligonucleotides attached thereto which are complementary to theoligonucleotides on at least one of the other types of nanoparticles.The nanoparticles of the aggregate probe are bound to each other as aresult of the hybridization of the oligonucleotides attached to them.Next, a type of nanoparticles having two types of oligonucleotidesattached thereto is provided. The first type of oligonucleotides has asequence complementary to a second portion of the sequence of saidnucleic acid, and the second type of oligonucleotides has a sequencecomplementary to a portion of the sequence of the oligonucleotidesattached to at least one of the types of nanoparticles of the coreprobe. The nucleic acid, the nanoparticles, the substrate and the coreprobe are contacted under conditions effective to allow hybridization ofsaid nucleic acid with the oligonucleotides on the nanoparticles andwith the oligonucleotides on the substrate and to allow hybridization ofthe oligonucleotides on the nanoparticles with the oligonucleotides onthe core probe, and a detectable change is observed.

[0018] Another embodiment of the method comprises providing a substratehaving oligonucleotides attached thereto, the oligonucleotides having asequence complementary to a first portion of the sequence of a nucleicacid to be detected. A core probe comprising at least two types ofnanoparticles is provided. Each type of nanoparticles hasoligonucleotides attached thereto which are complementary to theoligonucleotides on at least one other type of nanoparticles. Thenanoparticles of the aggregate probe are bound to each other as a resultof the hybridization of the oligonucleotides attached to them. A type oflinking oligonucleotides comprising a sequence complementary to a secondportion of the sequence of said nucleic acid and a sequencecomplementary to a portion of the sequence of the oligonucleotidesattached to at least one of the types of nanoparticles of the core probeis provided. The nucleic acid, the linking oligonucleotides, thesubstrate and the core probe are contacted under conditions effective toallow hybridization of said nucleic acid with the linkingoligonucleotides and with the oligonucleotides on the substrate and toallow hybridization of the oligonucleotides on the linkingoligonucleotides with the oligonucleotides on the core probe, and adetectable change is observed.

[0019] In yet another embodiment, the method comprises providingnanoparticles having oligonucleotides attached thereto and providing oneor more types of binding oligonucleotides. Each of the bindingoligonucleotides has two portions. The sequence of one portion iscomplementary to the sequence of one of the portions of the nucleicacid, and the sequence of the other portion is complementary to thesequence of the oligonucleotides on the nanoparticles. Thenanoparticle-oligonucleotide conjugates and the binding oligonucleotidesare contacted under conditions effective to allow hybridization of theoligonucleotides on the nanoparticles with the binding oligonucleotides.The nucleic acid and the binding oligonucleotides are contacted underconditions effective to allow hybridization of the bindingoligonucleotides with the nucleic acid. Then, a detectable change isobserved. The nanoparticle-oligonucleotide conjugates may be contactedwith the binding oligonucleotides prior to being contacted with thenucleic acid, or all three may be contacted simultaneously.

[0020] In another embodiment, the method comprises contacting a nucleicacid with at least two types of particles having oligonucleotidesattached thereto. The oligonucleotides on the first type of particleshave a sequence complementary to a first portion of the sequence of thenucleic acid and have energy donor molecules on the ends not attached tothe particles. The oligonucleotides on the second type of particles havea sequence complementary to a second portion of the sequence of thenucleic acid and have energy acceptor molecules on the ends not attachedto the particles. The contacting takes place under conditions effectiveto allow hybridization of the oligonucleotides on the particles with thenucleic acid, and a detectable change brought about by thishybridization is observed. The energy donor and acceptor molecules maybe fluorescent molecules.

[0021] In a further embodiment, the method comprises providing a type ofmicrospheres having oligonucleotides attached thereto. Theoligonucleotides have a sequence complementary to a first portion of thesequence of the nucleic acid and are labeled with a fluorescentmolecule. A type of nanoparticles having oligonucleotides attachedthereto and which produce a detectable change is also provided. Theseoligonucleotides have a sequence complementary to a second portion ofthe sequence of the nucleic acid. The nucleic acid is contacted with themicrospheres and the nanoparticles under conditions effective to allowhybridization of the oligonucleotides on the latex microspheres and onthe nanoparticles with the nucleic acid. Then, changes in fluorescence,another detectable change, or both are observed.

[0022] In another embodiment, the method comprises providing a firsttype of metallic or semiconductor nanoparticles having oligonucleotidesattached thereto. The oligonucleotides have a sequence complementary toa first portion of the sequence of the nucleic acid and are labeled witha fluorescent molecule. A second type of metallic or semiconductornanoparticles having oligonucleotides attached thereto is also provided.These oligonucleotides have a sequence complementary to a second portionof the sequence of the nucleic acid and are also labeled with afluorescent molecule. The nucleic acid is contacted with the two typesof nanoparticles under conditions effective to allow hybridization ofthe oligonucleotides on the two types of nanoparticles with the nucleicacid. Then, changes in fluorescence are observed.

[0023] In a further embodiment, the method comprises providing a type ofparticle having oligonucleotides attached thereto. The oligonucleotideshave a first portion and a second portion, both portions beingcomplementary to portions of the sequence of the nucleic acid. A type ofprobe oligonucleotides comprising a first portion and a second portionis also provided. The first portion has a sequence complementary to thefirst portion of the oligonucleotides attached to the particles, andboth portions are complementary to portions of the sequence of thenucleic acid. The probe oligonucleotides are also labeled with areporter molecule at one end. Then, the particles and the probeoligonucleotides are contacted under conditions effective to allow forhybridization of the oligonucleotides on the particles with the probeoligonucleotides to produce a satellite probe. Then, the satellite probeis contacted with the nucleic acid under conditions effective to providefor hybridization of the nucleic acid with the probe oligonucleotides.The particles are removed and the reporter molecule detected.

[0024] In yet another embodiment of the method of the invention, anucleic acid is detected by contacting the nucleic acid with a substratehaving oligonucleotides attached thereto. The oligonucleotides have asequence complementary to a first portion of the sequence of the nucleicacid. The oligonucleotides are located between a pair of electrodeslocated on the substrate. The contacting takes place under conditionseffective to allow hybridization of the oligonucleotides on thesubstrate with the nucleic acid. Then, the nucleic acid bound to thesubstrate, is contacted with a type of nanoparticles. The nanoparticlesare made of a material which can conduct electricity. The nanoparticleswill have one or more types of oligonucleotides attached to them, atleast one of the types of oligonucleotides having a sequencecomplementary to a second portion of the sequence of the nucleic acid.The contacting takes place under conditions effective to allowhybridization of the oligonucleotides on the nanoparticles with thenucleic acid. If the nucleic acid is present, a change in conductivitycan be detected. In a preferred embodiment, the substrate will have aplurality of pairs of electrodes located on it in an array to allow forthe detection of multiple portions of a single nucleic acid, thedetection of multiple different nucleic acids, or both. Each of thepairs of electrodes in the array will have a type of oligonucleotidesattached to the substrate between the two electrodes.

[0025] The invention further provides a method of detecting a nucleicacid whereint he method is performed on a substrate. The methodcomprises detecting the presence, quantity or both, of the nucleic acidwith an optical scanner.

[0026] The invention further provides kits for detecting nucleic acids.In one embodiment, the kit comprises at least one container, thecontainer holding at least two types of nanoparticles havingoligonucleotides attached thereto. The oligonucleotides on the firsttype of nanoparticles have a sequence complementary to the sequence of afirst portion of a nucleic acid. The oligonucleotides on the second typeof nanoparticles have a sequence complementary to the sequence of asecond portion of the nucleic acid.

[0027] Alternatively, the kit may comprise at least two containers. Thefirst container holds nanoparticles having oligonucleotides attachedthereto which have a sequence complementary to the sequence of a firstportion of a nucleic acid. The second container holds nanoparticleshaving oligonucleotides attached thereto which have a sequencecomplementary to the sequence of a second portion of the nucleic acid.

[0028] In a further embodiment, the kit comprises at least onecontainer. The container holds metallic or semiconductor nanoparticleshaving oligonucleotides attached thereto. The oligonucleotides have asequence complementary to portion of a nucleic acid and have fluorescentmolecules attached to the ends of the oligonucleotides not attached tothe nanoparticles.

[0029] In yet another embodiment, the kit comprises a substrate, thesubstrate having attached thereto nanoparticles, the nanoparticleshaving oligonucleotides attached thereto which have a sequencecomplementary to the sequence of a first portion of a nucleic acid. Thekit also includes a first container holding nanoparticles havingoligonucleotides attached thereto which have a sequence complementary tothe sequence of a second portion of the nucleic acid. The kit furtherincludes a second container holding a binding oligonucleotide having aselected sequence having at least two portions, the first portion beingcomplementary to at least a portion of the sequence of theoligonucleotides on the nanoparticles in the first container. The kitalso includes a third container holding nanoparticles havingoligonucleotides attached thereto, the oligonucleotides having asequence complementary to the sequence of a second portion of thebinding oligonucleotide.

[0030] In another embodiment, the kit comprises a substrate havingoligonucleotides attached thereto which have a sequence complementary tothe sequence of a first portion of a nucleic acid, a first containerholding nanoparticles having oligonucleotides attached thereto whichhave a sequence complementary to the sequence of a second portion of thenucleic acid, and a second container holding nanoparticles havingoligonucleotides attached thereto which have a sequence complementary toat least a portion of the oligonucleotides attached to the nanoparticlesin the first container.

[0031] In yet another embodiment, the kit comprises a substrate, a firstcontainer holding nanoparticles, a second container holding a first typeof oligonucleotides having a sequence complementary to the sequence of afirst portion of a nucleic acid, a third container holding a second typeof oligonucleotides having a sequence complementary to the sequence of asecond portion of the nucleic acid, and a fourth container holding athird type of oligonucleotides having a sequence complementary to atleast a portion of the sequence of the second type of oligonucleotides.

[0032] In a further embodiment, the kit comprises a substrate havingoligonucleotides attached thereto which have a sequence complementary tothe sequence of a first portion of a nucleic acid. The kit also includesa first container holding liposomes having oligonucleotides attachedthereto which have a sequence complementary to the sequence of a secondportion of the nucleic acid and a second container holding nanoparticleshaving at least a first type of oligonucleotides attached thereto, thefirst type of oligonucleotides having a hydrophobic group attached tothe end not attached to the nanoparticles so that the nanoparticles canbe attached to the liposomes by hydrophobic interactions. The kit mayfurther comprise a third container holding a second type ofnanoparticles having oligonucleotides attached thereto, theoligonucleotides having a sequence complementary to at least a portionof the sequence of a second type of oligonucleotides attached to thefirst type of nanoparticles. The second type of oligonucleotidesattached to the first type of nanoparticles have a sequencecomplementary to the sequence of the oligonucleotides on the second typeof nanoparticles.

[0033] In another embodiment, the kit comprises a substrate havingnanoparticles attached to it. The nanoparticles have oligonucleotidesattached to them which have a sequence complementary to the sequence ofa first portion of a nucleic acid. The kit also includes a firstcontainer holding an aggregate probe. The aggregated probe comprises atleast two types of nanoparticles having oligonucleotides attached tothem. The nanoparticles of the aggregate probe are bound to each otheras a result of the hybridization of some of the oligonucleotidesattached to each of them. At least one of the types of nanoparticles ofthe aggregate probe has oligonucleotides attached to it which have asequence complementary to a second portion of the sequence of thenucleic acid.

[0034] In yet another embodiment, the kit comprises a substrate havingoligonucleotides attached to it. The oligonucleotides have a sequencecomplementary to the sequence of a first portion of a nucleic acid. Thekit further includes a first container holding an aggregate probe. Theaggregate probe comprises at least two types of nanoparticles havingoligonucleotides attached to them. The nanoparticles of the aggregateprobe are bound to each other as a result of the hybridization of someof the oligonucleotides attached to each of them. At least one of thetypes of nanoparticles of the aggregate probe has oligonucleotidesattached thereto which have a sequence complementary to a second portionof the sequence of the nucleic acid.

[0035] In an additional embodiment, the kit comprises a substrate havingoligonucleotides attached to it and a first container holding anaggregate probe. The aggregate probe comprises at least two types ofnanoparticles having oligonucleotides attached to them. Thenanoparticles of the aggregate probe are bound to each other as a resultof the hybridization of some of the oligonucleotides attached to each ofthem. At least one of the types of nanoparticles of the aggregate probehas oligonucleotides attached to it which have a sequence complementaryto a first portion of the sequence of the nucleic acid. The kit alsoincludes a second container holding nanoparticles. The nanoparticleshave at least two types of oligonucleotides attached to them. The firsttype of oligonucleotides has a sequence complementary to a secondportion of the sequence of the nucleic acid. The second type ofoligonucleotides has a sequence complementary to at least a portion ofthe sequence of the oligonucleotides attached to the substrate.

[0036] In another embodiment, the kit comprises a substrate which hasoligonucleotides attached to it. The oligonucleotides have a sequencecomplementary to the sequence of a first portion of a nucleic acid. Thekit also comprises a first container holding liposomes havingoligonucleotides attached to them. The oligonucleotides have a sequencecomplementary to the sequence of a second portion of the nucleic acid.The kit further includes a second container holding an aggregate probecomprising at least two types of nanoparticles having oligonucleotidesattached to them. The nanoparticles of the aggregate probe are bound toeach other as a result of the hybridization of some of theoligonucleotides attached to each of them. At least one of the types ofnanoparticles of the aggregate probe has oligonucleotides attached to itwhich have a hydrophobic groups attached to the ends not attached to thenanoparticles.

[0037] In a further embodiment, the kit may comprise a first containerholding nanoparticles having oligonucleotides attached thereto. The kitalso includes one or more additional containers, each container holdinga binding oligonucleotide. Each binding oligonucleotide has a firstportion which has a sequence complementary to at least a portion of thesequence of oligonucleotides on the nanoparticles and a second portionwhich has a sequence complementary to the sequence of a portion of anucleic acid to be detected. The sequences of the second portions of thebinding oligonucleotides may be different as long as each sequence iscomplementary to a portion of the sequence of the nucleic acid to bedetected.

[0038] In another embodiment, the kit comprises a container holding onetype of nanoparticles having oligonucleotides attached thereto and oneor more types of binding oligonucleotides. Each of the types of bindingoligonucleotides has a sequence comprising at least two portions. Thefirst portion is complementary to the sequence of the oligonucleotideson the nanoparticles, whereby the binding oligonucleotides arehybridized to the oligonucleotides on the nanoparticles in thecontainer(s). The second portion is complementary to the sequence of aportion of the nucleic acid.

[0039] In another embodiment, kits may comprise one or two containersholding two types of particles. The first type of particles havingoligonucleotides attached thereto which have a sequence complementary tothe sequence of a first portion of a nucleic acid. The oligonucleotidesare labeled with an energy donor on the ends not attached to theparticles. The second type of particles having oligonucleotides attachedthereto which have a sequence complementary to the sequence of a secondportion of a nucleic acid. The oligonucleotides are labeled with anenergy acceptor on the ends not attached to the particles. The energydonors and acceptors may be fluorescent molecules.

[0040] In a further embodiment, the kit comprises a first containerholding nanoparticles having oligonucleotides attached thereto. The kitalso includes one or more additional containers, each container holdingbinding oligonucleotides. Each binding oligonucleotide has a firstportion which has a sequence complementary to at least a portion of thesequence of oligonucleotides on the nanoparticles and a second portionwhich has a sequence complementary to the sequence of a portion of anucleic acid to be detected. The sequences of the second portions of thebinding oligonucleotides may be different as long as each sequence iscomplementary to a portion of the sequence of the nucleic acid to bedetected.

[0041] In yet another embodiment, the kit comprises a container holdingone type of nanoparticles having oligonucleotides attached thereto andone or more types of binding oligonucleotides. Each of the types ofbinding oligonucleotides has a sequence comprising at least twoportions. The first portion is complementary to the sequence of theoligonucleotides on the nanoparticles, whereby the bindingoligonucleotides are hybridized to the oligonucleotides on thenanoparticles in the container(s). The second portion is complementaryto the sequence of a portion of the nucleic acid.

[0042] In another alternative embodiment, the kit comprises at leastthree containers. The first container holds nanoparticles. The secondcontainer holds a first oligonucleotide having a sequence complementaryto the sequence of a first portion of a nucleic acid. The thirdcontainer holds a second oligonucleotide having a sequence complementaryto the sequence of a second portion of the nucleic acid. The kit mayfurther comprise a fourth container holding a binding oligonucleotidehaving a selected sequence having at least two portions, the firstportion being complementary to at least a portion of the sequence of thesecond oligonucleotide, and a fifth container holding an oligonucleotidehaving a sequence complementary to the sequence of a second portion ofthe binding oligonucleotide.

[0043] In another embodiment, the kit comprises one or two containers,the container(s) holding two types of particles. The first type ofparticles having oligonucleotides attached thereto that have a sequencecomplementary to a first portion of the sequence of a nucleic acid andhave energy donor molecules attached to the ends not attached to thenanoparticles. The second type of particles having oligonucleotidesattached thereto that have a sequence complementary to a second portionof the sequence of a nucleic acid and have energy acceptor moleculesattached to the ends not attached to the nanoparticles. The energydonors and acceptors may be fluorescent molecules.

[0044] In a further embodiment, the kit comprises a first containerholding a type of microspheres having oligonucleotides attached thereto.The oligonucleotides have a sequence complementary to a first portion ofthe sequence of a nucleic acid and are labeled with a fluorescentmolecule. The kit also comprises a second container holding a type ofnanoparticles having oligonucleotides attached thereto. Theoligonucleotides have a sequence complementary to a second portion ofthe sequence of the nucleic acid.

[0045] In another embodiment, the kit comprises a first containerholding a first type of metallic or semiconductor nanoparticles havingoligonucleotides attached thereto. The oligonucleotides have a sequencecomplementary to a first portion of the sequence of a nucleic acid andare labeled with a fluorescent molecule. The kit also comprises a secondcontainer holding a second type of metallic or semiconductornanoparticles having oligonucleotides attached thereto. Theseoligonucleotides have a sequence complementary to a second portion ofthe sequence of a nucleic acid and are labeled with a fluorescentmolecule.

[0046] In another embodiment, the kit comprises a container holding anaggregate probe. The aggregate probe comprises at least two types ofnanoparticles having oligonucleotides attached to them. Thenanoparticles of the aggregate probe are bound to each other as a resultof the hybridization of some of the oligonucleotides attached to each ofthem. At least one of the types of nanoparticles of the aggregate probehas oligonucleotides attached to it which have a sequence complementaryto a portion of the sequence of a nucleic acid.

[0047] In an additional embodiment, the kit comprises a containerholding an aggregate probe. The aggregate probe comprises at least twotypes of nanoparticles having oligonucleotides attached to them. Thenanoparticles of the aggregate probe are bound to each other as a resultof the hybridization of some of the oligonucleotides attached to each ofthem. At least one of the types of nanoparticles of the aggregate probehas oligonucleotides attached to it which have a hydrophobic groupattached to the end not attached to the nanoparticles.

[0048] In a further embodiment, the kit comprises a container holding asatellite probe. The satellite probe comprises a particle havingattached thereto oligonucleotides. The oligonucleotides have a firstportion and a second portion, both portions having sequencescomplementary to portions of the sequence of a nucleic acid. Thesatellite probe also comprises probe oligonucleotides hybridized to theoligonucleotides attached to the nanoparticles. The probeoligonucleotides have a first portion and a second portion. The firstportion has a sequence complementary to the sequence of the firstportion of the oligonucleotides attached to the particles, and bothportions have sequences complementary to portions of the sequence of thenucleic acid. The probe oligonucleotides also have a reporter moleculeattached to one end.

[0049] In another embodiment, the kit comprising a container holding acore probe, the core probe comprising at least two types ofnanoparticles having oligonucleotides attached thereto, thenanoparticles of the core probe being bound to each other as a result ofthe hybridization of some of the oligonucleotides attached to them.

[0050] In yet another embodiment, the kit comprises a substrate havingattached to it at least one pair of electrodes with oligonucleotidesattached to the substrate between the electrodes. The oligonucleotideshave a sequence complementary to a first portion of the sequence of anucleic acid to be detected.

[0051] The invention also provides the satellite probe, an aggregateprobe and a core probe.

[0052] The invention further provides a substrate having nanoparticlesattached thereto. The nanoparticles may have oligonucleotides attachedthereto which have a sequence complementary to the sequence of a firstportion of a nucleic acid.

[0053] The invention also provides a metallic or semiconductornanoparticle having oligonucleotides attached thereto. Theoligonucleotides are labeled with fluorescent molecules at the ends notattached to the nanoparticle.

[0054] The invention further provides a method of nanofabrication. Themethod comprises providing at least one type of linking oligonucleotidehaving a selected sequence, the sequence of each type of linkingoligonucleotide having at least two portions. The method furthercomprises providing one or more types of nanoparticles havingoligonucleotides attached thereto, the oligonucleotides on each type ofnanoparticles having a sequence complementary to a portion of thesequence of a linking oligonucleotide. The linking oligonucleotides andnanoparticles are contacted under conditions effective to allowhybridization of the oligonucleotides on the nanoparticles to thelinking oligonucleotides so that a desired nanomaterials ornanostructure is formed.

[0055] The invention provides another method of nanofabrication. Thismethod comprises providing at least two types of nanoparticles havingoligonucleotides attached thereto. The oligonucleotides on the firsttype of nanoparticles have a sequence complementary to that of theoligonucleotides on the second type of nanoparticles. Theoligonucleotides on the second type of nanoparticles have a sequencecomplementary to that of the oligonucleotides on the first type ofnanoparticle-oligonucleotide conjugates. The first and second types ofnanoparticles are contacted under conditions effective to allowhybridization of the oligonucleotides on the nanoparticles to each otherso that a desired nanomaterials or nanostructure is formed.

[0056] The invention further provides nanomaterials or nanostructurescomposed of nanoparticles having oligonucleotides attached thereto, thenanoparticles being held together by oligonucleotide connectors.

[0057] The invention also provides a composition comprising at least twotypes of nanoparticles having oligonucleotides attached thereto. Theoligonucleotides on the first type of nanoparticles have a sequencecomplementary to the sequence of a first portion of a nucleic acid or alinking oligonucleotide. The oligonucleotides on the second type ofnanoparticles have a sequence complementary to the sequence of a secondportion of the nucleic acid or linking oligonucleotide.

[0058] The invention further provides an assembly of containerscomprising a first container holding nanoparticles havingoligonucleotides attached thereto, and a second container holdingnanoparticles having oligonucleotides attached thereto. Theoligonucleotides attached to the nanoparticles in the first containerhave a sequence complementary to that of the oligonucleotides attachedto the nanoparticles in the second container. The oligonucleotidesattached to the nanoparticles in the second container have a sequencecomplementary to that of the oligonucleotides attached to thenanoparticles in the first container.

[0059] The invention also provides a nanoparticle having a plurality ofdifferent oligonucleotides attached to it.

[0060] The invention further provides a method of separating a selectednucleic acid having at least two portions from other nucleic acids. Themethod comprises providing one or more types of nanoparticles havingoligonucleotides attached thereto, the oligonucleotides on each of thetypes of nanoparticles having a sequence complementary to the sequenceof one of the portions of the selected nucleic acid. The selectednucleic acid and other nucleic acids are contacted with thenanoparticles under conditions effective to allow hybridization of theoligonucleotides on the nanoparticles with the selected nucleic acid sothat the nanoparticles hybridized to the selected nucleic acid aggregateand precipitate.

[0061] In addition, the invention provides methods of making uniquenanoparticle-oligonucleotide conjugates. The first such method comprisesbinding oligonucleotides to charged nanoparticles to produce stablenanoparticle-oligonucleotide conjugates. To do so, oligonucleotideshaving covalently bound thereto a moiety comprising a functional groupwhich can bind to the nanoparticles are contacted with the nanoparticlesin water for a time sufficient to allow at least some of theoligonucleotides to bind to the nanoparticles by means of the functionalgroups. Next, at least one salt is added to the water to form a saltsolution. to The ionic strength of the salt solution must be sufficientto overcome at least partially the electrostatic repulsion of theoligonucleotides from each other and, either the electrostaticattraction of the negatively-charged oligonucleotides forpositively-charged nanoparticles, or the electrostatic repulsion of thenegatively-charged oligonucleotides from negatively-chargednanoparticles. After adding the salt, the oligonucleotides andnanoparticles are incubated in the salt solution for an additionalperiod of time sufficient to allow sufficient additionaloligonucleotides to bind to the nanoparticles to produce the stablenanoparticle-oligonucleotide conjugates. The invention also includes thestable nanoparticle-oligonucleotide conjugates, methods of using theconjugates to detect and separate nucleic acids, kits comprising theconjugates, methods of nanofabrication using the conjugates, andnanomaterials and nanostructures comprising the conjugates.

[0062] The invention provides another method of binding oligonucleotidesto nanoparticles to produce nanoparticle-oligonucleotide conjugates. Themethod comprises providing oligonucleotides, the oligonucleotidescomprising a type of recognition oligonucleotides and a type of diluentoligonucleotides. The oligonucleotides and the nanoparticles arecontacted under conditions effective to allow at least some of each ofthe types of oligonucleotides to bind to the nanoparticles to producethe conjugates. The invention also includes thenanoparticle-oligonucleotide conjugates produced by this method, methodsof using the conjugates to detect and separate nucleic acids, kitscomprising the conjugates, methods of nanofabrication using theconjugates, and nanomaterials and nanostructures comprising theconjugates. “Recognition oligonucleotides” are oligonucleotides whichcomprise a sequence complementary to at least a portion of the sequenceof a nucleic acid or oligonucleotide target. “Diluent oligonucleotides”may have any sequence which does not interfere with the ability of therecognition oligonucleotides to be bound to the nanoparticles or to bindto their targets.

[0063] The invention provides yet another method of bindingoligonucleotides to nanoparticles to producenanoparticle-oligonucleotide conjugates. The method comprises providingoligonucleotides, the oligonucleotides comprising at least one type ofrecognition oligonucleotides. The recognition oligonucleotides comprisea recognition portion and a spacer portion. The recognition portion ofthe recognition oligonucleotides has a sequence complementary to atleast one portion of the sequence of a nucleic acid or oligonucleotidetarget. The spacer portion of the recognition oligonucleotide isdesigned so that it can bind to the nanoparticles. As a result of thebinding of the spacer portion of the recognition oligonucleotide to thenanoparticles, the recognition portion is spaced away from the surfaceof the nanoparticles and is more accessible for hybridization with itstarget. To make the conjugates, the oligonucleotides, including therecognition oligonucleotides, and the nanoparticles are contacted underconditions effective allow at least some of the recognitionoligonucleotides to bind to the nanoparticles. The invention alsoincludes the nanoparticle-oligonucleotide conjugates produced by thismethod, methods of using the conjugates to detect and separate nucleicacids, kits comprising the conjugates, methods of nanofabrication usingthe conjugates, and nanomaterials and nanostructures comprising theconjugates.

[0064] As used herein, a “type of oligonucleotides” refers to aplurality of oligonucleotide molecules having the same sequence. A “typeof” nanoparticles, conjugates, particles, latex microspheres, etc.having oligonucleotides attached thereto refers to a plurality of thatitem having the same type(s) of oligonucleotides attached to them.“Nanoparticles having oligonucleotides attached thereto” are alsosometimes referred to as “nanoparticle-oligonucleotide conjugates” or,in the case of the detection methods of the invention,“nanoparticle-oligonucleotide probes,” “nanoparticle probes,” or just“probes.”

BRIEF DESCRIPTION OF THE DRAWINGS

[0065]FIG. 1: Schematic diagram illustrating the formation ofnanoparticle aggregates by combining nanoparticles having complementaryoligonucleotides attached to them, the nanoparticles being held togetherin the aggregates as a result of the hybridization of the complementaryoligonucleotides. X represents any covalent anchor (such as—S(CH₂)₃OP(O)(O⁻)—, where S is joined to a gold nanoparticle). For thesake of simplicity in FIG. 1 and some subsequent figures, only oneoligonucleotide is shown to be attached to each particle but, in fact,each particle has several oligonucleotides attached to it. Also, it isimportant to note that in FIG. 1 and subsequent figures, the relativesizes of the gold nanoparticles and the oligonucleotides are not drawnto scale.

[0066]FIG. 2: Schematic diagram illustrating a system for detectingnucleic acid using nanoparticles having oligonucleotides attachedthereto. The oligonucleotides on the two nanoparticles have sequencescomplementary to two different portions of the single-stranded DNAshown. As a consequence, they hybridize to the DNA producing detectablechanges (forming aggregates and producing a color change).

[0067]FIG. 3: Schematic diagram of a variation of the system shown inFIG. 2. The oligonucleotides on the two nanoparticles have sequencescomplementary to two different portions of the single-stranded DNA shownwhich are separated by a third portion which is not complementary to theoligonucleotides on the nanoparticles. Also shown is an optional filleroligonucleotide which can be used to hybridize with the noncomplementaryportion of the single-stranded DNA. When the DNA, nanoparticles andfiller oligonucleotides are combined, the nanoparticles aggregate, withthe formation of nicked, double-stranded oligonucleotide connectors.

[0068]FIG. 4: Schematic diagram illustrating reversible aggregation ofnanoparticles having oligonucleotides attached thereto as a result ofhybridization and de-hybridization with a linking oligonucleotide. Theillustrated linking oligonucleotide is a double-stranded DNA havingoverhanging termini (sticky ends) which are complementary to theoligonucleotides attached to the nanoparticles.

[0069]FIG. 5: Schematic diagram illustrating the formation ofnanoparticle aggregates by combining nanoparticles havingoligonucleotides attached thereto with linking oligonucleotides havingsequences complementary to the oligonucleotides attached to thenanoparticles.

[0070]FIG. 6: Cuvettes containing two types of gold colloids, eachhaving a different oligonucleotide attached thereto and a linkingdouble-stranded oligonucleotide with sticky ends complementary to theoligonucleotides attached to the nanoparticles (see FIG. 4). CuvetteA—at 80° C., which is above the Tm of the linking DNA; de-hybridized(thermally denatured). The color is dark red. Cuvette B—after cooling toroom temperature, which is belowthe Tm of the linking DNA; hybridizationhas taken place, and the nanoparticles have aggregated, but theaggregates have not precipitated. The color is purple. Cuvette C—afteris several hours at room temperature, the aggregated nanoparticles havesettled to the bottom of the cuvette. The solution is clear, and theprecipitate is pinkish gray. Heating B or C will result in A.

[0071]FIG. 7: A graph of absorbance versus wavelength in nm showingchanges in absorbance when gold nanoparticles having oligonucleotidesattached thereto aggregate due to hybridization with linkingoligonucleotides upon lowering of the temperature, as illustrated inFIG. 4.

[0072] FIGS. 8A-B: FIG. 8A is a graph of change in absorbance versustemperature/time for the system illustrated in FIG. 4. At lowtemperatures, gold nanoparticles having oligonucleotides attachedthereto aggregate due to hybridization with linking oligonucleotides(see FIG. 4). At high temperature (80° C.), the nanoparticles arede-hybridized. Changing the temperature over time shows that this is areversible process.

[0073]FIG. 8B is a graph of change in absorbance versus temperature/timeperformed in the same manner using an aqueous solution of unmodifiedgold nanoparticles. The reversible changes seen in FIG. 8A are notobserved.

[0074] FIGS. 9A-B: Transmission Electron Microscope (TEM) images.

[0075]FIG. 9A is a TEM image of aggregated gold nanoparticles heldtogether by hybridization of the oligonucleotides on the goldnanoparticles with linking oligonucleotides.

[0076]FIG. 9B is a TEM image of a two-dimensional aggregate showing theordering of the linked nanoparticles.

[0077]FIG. 10: Schematic diagram illustrating the formation ofthermally-stable triple-stranded oligonucleotide connectors betweennanoparticles having the pyrimidine:purine:pyrimidine motif. Suchtriple-stranded connectors are stiffer than double-stranded connectors.In FIG. 10, one nanoparticle has an oligonucleotide attached to it whichis composed of all purines, and the other nanoparticle has anoligonucleotide attached to it which is composed of all pyrimidines. Thethird oligonucleotide for forming the triple-stranded connector (notattached to a nanoparticle) is composed of pyrimidines.

[0078]FIG. 11: Schematic diagram illustrating the formation ofnanoparticle aggregates by combining nanoparticles having complementaryoligonucleotides attached to them, the nanoparticles being held togetherin the aggregates as a result of the hybridization of the complementaryoligonucleotides. In FIG. 11, the circles represent the nanoparticles,the formulas are oligonucleotide sequences, and s is the thio-alkyllinker. The multiple oligonucleotides on the two types of nanoparticlescan hybridize to each other, leading to the formation of an aggregatestructure.

[0079] FIGS. 12A-F: Schematic diagrams illustrating systems fordetecting nucleic acid using nanoparticles having oligonucleotidesattached thereto. Oligonucleotide-nanoparticle conjugates 1 and 2 andsingle-stranded oligonucleotide targets 3, 4, 5, 6 and 7 areillustrated. The circles represent the nanoparticles, the formulas areoligonucleotide sequences, and the dotted and dashed lines representconnecting links of nucleotide.

[0080] FIGS. 13A-B: Schematic diagrams illustrating systems fordetecting DNA (analyte DNA) using nanoparticles and a transparentsubstrate.

[0081] FIGS. 14A-B: FIG. 14A is a graph of absorbance versus wavelengthin nm showing changes in absorbance when gold nanoparticles havingoligonucleotides attached thereto (one population of which is insolution and one population of which is attached to a transparentsubstrate as illustrated in FIG. 13B) aggregate due to hybridizationwith linking oligonucleotides.

[0082]FIG. 14B a graph of change in absorbance for the hybridized systemreferred to in FIG. 14A as the temperature is increased (melted).

[0083] FIGS. 15A-G: Schematic diagrams illustrating systems fordetecting nucleic acid using nanoparticles having oligonucleotidesattached thereto. Oligonucleotide-nanoparticle conjugates 1 and 2 andsingle-stranded oligonucleotide targets 3, 4, 5, 6, 7 and 8 areillustrated. The circles represent the nanoparticles, the formulas areoligonucleotide sequences, and S represents the thio-alkyl linker.

[0084] FIGS. 16A-C: Schematic diagrams illustrating systems fordetecting nucleic acid using nanoparticles having oligonucleotidesattached thereto. Oligonucleotide-nanoparticle conjugates 1 and 2,single-stranded oligonucleotide targets of different lengths, and filleroligonucleotides of different lengths are illustrated. The circlesrepresent the nanoparticles, the formulas are oligonucleotide sequences,and S represents the thio-alkyl linker.

[0085] FIGS. 17A-E: Schematic diagrams illustratingnanoparticle-oligonucleotide conjugates and systems for detectingnucleic acid using nanoparticles having oligonucleotides attachedthereto. The circles represent the nanoparticles, the straight linesrepresent oligonucleotide chains (bases not shown), two closely-spacedparallel lines represent duplex segments, and the small letters indicatespecific nucleotide sequences (a is complementary to a′, b iscomplementary to b′, etc.).

[0086]FIG. 18: Schematic diagram illustrating a system for detectingnucleic acid using liposomes (large double circle), nanoparticles (smallopen circles) and a transparent substrate. The filled-in squaresrepresent cholesteryl groups, the squiggles represent oligonucleotides,and the ladders represent double-stranded (hybridized) oligonucleotides.

[0087] FIGS. 19A-B: FIG. 19A is a graph of absorbance versus wavelengthin nm showing changes in absorbance when goldnanoparticle-oligonucleotide conjugates assemble in multiple layers on atransparent substrate as illustrated in FIG. 13A.

[0088]FIG. 19B is a graph of change in absorbance for the hybridizedsystem referred to in FIG. 19A as the temperature is increased (melted).

[0089] FIGS. 20A-B: Illustrations of schemes using fluorescent-labeledoligonucleotides attached to metallic or semiconductor quenchingnanoparticles (FIG. 20A) or to non-metallic, non-semiconductor particles(FIG. 20B).

[0090]FIG. 21: Schematic diagram illustrating a system for detectingtarget nucleic acid using gold nanoparticles having oligonucleotidesattached thereto and latex microspheres having fluorescently-labeledoligonucleotides attached thereto. The small, closed, dark circlesrepresent the nanoparticles, the large, open circles represent the latexmicrospheres, and the large oval represents a microporous membrane.

[0091]FIG. 22: Schematic diagram illustrating a system for detectingtarget nucleic acid using two types of fluorescently-labeledoligonucleotide-nanoparticle conjugates. The closed circles representthe nanoparticles, and the large oval represents a microporous membrane.

[0092]FIG. 23: Sequences of materials utilized in an assay for AnthraxProtective Antigen (see Example 12).

[0093]FIG. 24: Schematic diagram illustrating a system for detectingtarget nucleic acid using a “satellite probe” which comprises magneticnanoparticles (dark spheres) having oligonucleotides (straight lines)attached to them, probe oligonucleotides (straight lines) hybridized tothe oligonucleotides attached to the nanoparticles, the probeoligonucleotides being labeled with a reporter group (open rectangularbox). A, B, C, A′, B′, and C′ represent specific nucleotide sequences,with A, B and C being complementary to A′, B′ and C′, respectively.

[0094] FIGS. 25A-B: Schematic diagrams illustrating systems fordetecting DNA using nanoparticles and a transparent substrate. In thesefigures, a, b and c refer to different oligonucleotide sequences, anda′, b′ and c′ refer to oligonucleotide sequences complementary to a, band c, respectively.

[0095]FIG. 26: Schematic diagram illustrating systems for formingassemblies of CdSe/ZnS core/shell quantum dots (QD).

[0096] FIGS. 27A-D: FIG. 27A shows fluorescence spectra comparingdispersed and aggregated QDs, with an excitation at 400 nm. The sampleswere prepared identically, except for the addition of complementary“linker” DNA to one and an equal volume and concentration ofnon-complementary DNA to the other.

[0097]FIG. 27B shows UV-Visible spectra of QD/QD assemblies at differenttemperatures before, during and after “melting”.

[0098]FIG. 27C shows high resolution TEM image of a portion of a hybridgold/QD assembly. The lattice fringes of the QDs, which resemblefingerprints, appear near each gold nanoparticle.

[0099]FIG. 27D shows UV-Visible spectra of hybrid gold/QD assemblies atdifferent temperatures before, during and after “melting”. The insets inFIGS. 27B and 27D display temperature versus extinction profiles for thethermal denaturation of the assemblies. Denturation experiments wereconducted in 0.3 M NaCl, 10 mM phosphate buffer (pH 7), 0.01% sodiumazide with 13 nm gold nanoparticles and/or ˜4 nm CdSe/ZnS core/shellQDs.

[0100] FIGS. 28A-E: Schematic diagrams illustrating the preparation ofcore probes, aggregate probes and systems for detecting DNA using theseprobes. In these figures, a, b, c and d refer to differentoligonucleotide sequences, and a′, b′, c′ and d′ refer tooligonucleotide sequences complementary to a, b, c and d, respectively.

[0101]FIG. 29: Graph of fractional displacement of oligonucleotides bymercaptoethanol from nanoparticles (closed circles) or gold thin films(open squares) to which the oligonucleotides had been attached.

[0102]FIG. 30: Graph of surface coverages of recognitionoligonucleotides on nanoparticles obtained for different ratios ofrecognition:diluent oligonucleotides used in the preparation of thenanoparticle-oligonucleotide conjugates.

[0103]FIG. 31: Graph of surface coverages of hybridized complementaryoligonucleotides versus different surface coverages of recognitionoligonucleotides on nanoparticles.

[0104]FIG. 32: Schematic diagram illustrating system for detecting atarget DNA in a four-element array on a substrate usingnanoparticle-oligonucleotide conjugates and amplification with silverstaining.

[0105]FIG. 33: Images obtained with a flatbed scanner of 7 mm×13 mmoligonucleotide-functionalized float glass slides. (A) Slide beforehybridization of DNA target and gold nanoparticle-oligonucleotideindicator conjugate. (B) Slide A after hybridization of 10 nM target DNAand 5 nM nanoparticle-oligonucleotide indicator conjugate. A pink colorwas imparted by attached, red 13 nm diameter gold nanoparticles. (C)Slide B after exposure to silver amplification solution for 5 minutes.(D) Same as (A). (E) Slide D after hybridization of 100 pM target and 5nM nanoparticle-oligonucleotide indicator conjugate. The absorbance ofthe nanoparticle layer was too low to be observed with the naked eye orflatbed scanner. (F) Slide E after exposure to silver amplificationsolution for 5 minutes. Note that slide F is much lighter than slide C,indicating lower target concentration. (G) Control slide, exposed to 5nM nanoparticle-oligonucleotide indicator conjugate and exposed tosilver amplification solution for 5 minutes. No darkening of the slidewas observed.

[0106]FIG. 33: Graph of greyscale (optical density) ofoligonucleotide-functionalized glass surface exposed to varyingconcentrations of target DNA, followed by 5 nM gold ofnanoparticle-oligonucleotide indicator conjugates and silveramplification for 5 minutes.

[0107] FIGS. 35A-B: Graphs of percent hybridized label versustemperature showing dissociation of fluorophore-labeled (FIG. 35A) andnanoparticle-labeled (FIG. 35B) targets from anoligonucleotide-functionalized glass surface. Measurements were made bymeasuring fluorescence (FIG. 35A) or absorbance (FIG. 35B) ofdissociated label in the solution above the glass surface. The lineslabeled “b” show the dissociation curves for perfectly matchedoligonucleotides on the glass, and the lines labeled “r” show curves formismatched oligonucleotides (a one-base mismatch) on the glass. Verticallines in the graphs illustrate the fraction of target dissociated at agiven temperature (halfway between the melting temperatures T_(m) ofeach curve) for each measurement, and the expected selectivity ofsequence identification for fluorophore- and nanoparticle-based genechips. Fluorescence (FIG. 35A): complement (69%)/mismatch (38%)=1.8:1.Absorbance (FIG. 35B): complement (85%)/mismatch (14%)=6:1. The breadthof the fluorophore-labeled curves (FIG. 35A) is characteristic of thedissociation of fluorophore-labeled targets from gene chips (Forman etal., in Molecular Modeling of Nucleic Acids, Leontis et al., eds., (ACSSymposium Series 682, American Chemical Society, Washington D.C., 1998),pages 206-228).

[0108] FIGS. 36A-B: Images of model oligonucleotide arrays challengedwith synthetic target and fluorescent-labeled (FIG. 36A) ornanoparticle-labeled (FIG. 36B) nanoparticle-oligonucleotide conjugateprobes. C, A, T, and G represent spots (elements) on the array where asingle base change has been made in the oligonucleotide attached to thesubstrate to give a perfect match with the target (base A) or a singlebase mismatch (base C, T or G in place of the perfect match with baseA). The greyscale ratio for elements C:A:T:G is 9:37:9:11 for FIG. 36Aand 3:62:7:34 for FIG. 36B.

[0109]FIG. 37: Schematic diagram illustrating system for formingaggregates (A) or is layers (B) of nanoparticles (a and b) linked by alinking nucleic acid (3).

[0110]FIG. 38A: UV-visible spectra of alternating layers of goldnanoparticles a and b (see FIG. 37) hybridized to anoligonucleotide-functionalized glass microscope slide via thecomplementary linker 3. The spectra are for assemblies with 1 (a,λ_(max)=524 nm), 2 (b, λ_(max)=529 nm), 3 (c, λ_(max)=532 nm), 4 (d,λ_(max)=534 nm) or 5 (e, λ_(max)=534 nm) layers. These spectra weremeasured directly through the slide.

[0111]FIG. 38B: Graph of absorbance for nanoparticle assemblies (seeFIG. 38A) at λ_(max) with increasing numbers of layers.

[0112] FIGS. 39A-F: FIG. 39A: FE-SEM of one layer ofoligonucleotide-functionalized gold nanoparticles cohybridized with DNAlinker to an oligonucleotide-functionalized, 25 conductiveindium-tin-oxide (ITO) slide (prepared in the same way asoligonucleotide-funcationalized glass slide). The visible absorbancespectrum of this slide was identical to FIG. 38A, indicating thatfunctionalization and nanoparticle coverage on ITO is similar to that onglass. The average density of counted nanoparticles from 10 such imageswas approximately 800 nanoparticles/μm². FIG. 39B: FE-SEM image of twolayers of nanoparticles on the ITO slide. The average density of countednanoparticles from 10 such images was approximately 2800 particles/μm².FIG. 39C: Absorbance at 260 nm (A₂₆₀) showing dissociation of a 0.5 μMsolution of the oligonucleotide duplex (1+2+3; see FIG. 37, A) to singlestrands in 0.3 M NaCl, 10 mM phosphate buffer solution (pH 7). FIGS.39D-F: Absorbance at 260 nm (A₂₆₀) showing dissociation of 1 layer (FIG.39D), 4 layers (FIG. 39E) and 10 layers (FIG. 39F) ofoligonucleotide-functionalized gold nanoparticles from glass slidesimmersed in 0.3 M NaCl, 10 mM phosphate buffer solution. Meltingprofiles were obtained by measuring the decreasing absorption at 520 nm(A₅₂₀) through the slides with increasing temperature. In each of FIGS.39D-F, the insets show the first derivatives of the measureddissociation curves. FWHM of these curves were (FIG. 39C inset) 13.2°C., (FIG. 39D inset) 5.6° C., (FIG. 39E inset) 3.2° C., and (FIG. 39Finset) 2.9° C.

[0113]FIG. 40: Schematic diagram illustrating system used to measure theelectrical properties of gold nanoparticle assemblies linked by DNA. Forsimplicity, only one hybridization event is drawn.

[0114]FIG. 41: Schematic diagram illustrating a method of detectingnucleic acid using gold electrodes and gold nanoparticles.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

[0115] Nanoparticles useful in the practice of the invention includemetal (e.g., gold, silver, copper and platinum), semiconductor (e.g.,CdSe, CdS, and CdS or CdSe coated with ZnS) and magnetic (e.g.,ferromagnetite) colloidal materials. Other nanoparticles useful in thepractice of the invention include ZnS, ZnO, TiO₂, AgI, AgBr, HgI₂, PbS,PbSe, ZnTe, CdTe, In₂S₃, In₂Se₃, Cd₃P₂, Cd₃As₂, InAs, and GaAs. The sizeof the nanoparticles is preferably from about 5 nm to about 150 nm (meandiameter), more preferably from about 5 to about 50 nm, most preferablyfrom about 10 to about 30 nm. The nanoparticles may also be rods.

[0116] Methods of making metal, semiconductor and magnetic nanoparticlesare well-known in the art. See, e.g., Schmid, G. (ed.) Clusters andColloids (VCH, Weinheim, 1994); Hayat, M. A. (ed.) Colloidal Gold:Principles, Methods, and Applications (Academic Press, San Diego, 1991);Massart, R., IEEE Taransactions On Magnetics, 17, 1247 (1981); Ahmadi,T. S. et al., Science, 272,1924 (1996); Henglein, A. et al., J. Phys.Chem., 99,14129 (1995); Curtis, A. C., et al., Angew. Chem. Int. Ed.Engl., 27, 1530 (1988).

[0117] Methods of making ZnS, ZnO, TiO₂, AgI, AgBr, HgI₂, PbS, PbSe,ZnTe, CdTe, In2S3, In₂Se₃, Cd₃P₂, Cd₃As₂, InAs, and GaAs nanoparticlesare also known in the art. See, e.g., Weller, Angew. Chem. Int. Ed.Engl., 32, 41 (1993); Henglein, Top. Curr. Chem., 143, 113 (1988);Henglein, Chem. Rev., 89, 1861 (1989); Brus, Appl. Phys. A., 53, 465(1991); Bahncmann, in Photochemical Conversion and Storage of SolarEnergy (eds. Pelizetti and Schiavello 1991), page 251; Wang and Herron,J. Phys. Chem., 95, 525 (1991); Olshavsky et al., J. Am. Chem. Soc.,112, 9438 (1990); Ushida et al., J. Phys. Chem., 95, 5382 (1992).

[0118] Suitable nanoparticles are also commercially available from,e.g., Ted Pella, Inc. (gold), Amersham Corporation (gold) andNanoprobes, Inc. (gold).

[0119] Presently preferred for use in detecting nucleic acids are goldnanoparticles. Gold colloidal particles have high extinctioncoefficients for the bands that give rise to their beautiful colors.These intense colors change with particle size, concentration,interparticle distance, and extent of aggregation and shape (geometry)of the aggregates, making these materials particularly attractive forcolorimetric assays. For instance, hybridization of oligonucleotidesattached to gold nanoparticles with oligonucleotides and nucleic acidsresults in an immediate color change visible to the naked eye (see,e.g., the Examples).

[0120] Gold nanoparticles are also presently preferred for use innanofabrication for the same reasons given above and because of theirstability, ease of imaging by electron microscopy, andwell-characterized modification with thiol functionalities (see below).Also preferred for use in nanofabrication are semiconductornanoparticles because of their unique electronic and luminescentproperties.

[0121] The nanoparticles, the oligonucleotides or both arefunctionalized in order to attach the oligonucleotides to thenanoparticles. Such methods are known in the art. For instance,oligonucleotides functionalized with alkanethiols at their 3′-termini or5′-termini readily attach to gold nanoparticles. See Whitesides,Proceedings of the Robert A. Welch Foundation 39th Conference OnChemical Research Nanophase Chemistry, Houston, Tex., pages 109-121(1995). See also, Mucic et al. Chem. Commun. 555-557 (1996) (describes amethod of attaching 3′ thiol DNA to flat gold surfaces; this method canbe used to attach oligonucleotides to nanoparticles). The alkanethiolmethod can also be used to attach oligonucleotides to other metal,semiconductor and magnetic colloids and to the other nanoparticleslisted above. Other functional groups for attaching oligonucleotides tosolid surfaces include phosphorothioate groups (see, e.g., U.S. Pat. No.5,472,881 for the binding of oligonucleotide-phosphorothioates to goldsurfaces), substituted alkylsiloxanes (see, e.g. Burwell, ChemicalTechnology, 4, 370-377 (1974) and Matteucci and Caruthers, J. Am. Chem.Soc., 103,3185-3191 (1981) for binding of oligonucleotides to silica andglass surfaces, and Grabar et al., Anal. Chem., 67,735-743 for bindingof aminoalkylsiloxanes and for similar binding ofmercaptoaklylsiloxanes). Oligonucleotides terminated with a 5′thionucleoside or a 3′ thionucleoside may also be used for attachingoligonucleotides to solid surfaces. The following references describeother methods which may be employed to attached oligonucleotides tonanoparticles: Nuzzo et al., J. Am. Chem. Soc., 109, 2358 (1987)(disulfides on gold); Allara and Nuzzo, Langmuir, 1, 45 (1985)(carboxylic acids on aluminum); Allara and Tompkins, J. ColloidInterface Sci., 49, 410421 (1974) (carboxylic acids on copper); Iler,The Chemistry Of Silica, Chapter 6, (Wiley 1979) (carboxylic acids onsilica); Timmons and Zisman, J. Phys. Chem., 69, 984-990 (1965)(carboxylic acids on platinum); Soriaga and Hubbard, J. Am. Chem. Soc.,104, 3937 (1982) (aromatic ring compounds on platinum); Hubbard, Acc.Chem. Res., 13,177 (1980) (sulfolanes, sulfoxides and otherfunctionalized solvents on platinum); Hickman et al., J. Am. Chem. Soc.,11 1, 7271 (1989) (isonitriles on platinum); Maoz and Sagiv, Langmuir,3, 1045 (1987) (silanes on silica); Maoz and Sagiv, Langmuir, 3, 1034(1987) (silanes on silica); Wasserman et al., Langmuir, 5, 1074 (1989)(silanes on silica); Eltekova and Eltekov, Langmuir, 3,951 (1987)(aromatic carboxylic acids, aldehydes, alcohols and methoxy groups ontitanium dioxide and silica); Lec et al., J. Phys. Chem., 92, 2597(1988) (rigid phosphates on metals).

[0122] Each nanoparticle will have a plurality of oligonucleotidesattached to it. As a result, each nanoparticle-oligonucleotide conjugatecan bind to a plurality of oligonucleotides or nucleic acids having thecomplementary sequence.

[0123] Oligonucleotides of defined sequences are used for a variety ofpurposes in the practice of the invention. Methods of makingoligonucleotides of a predetermined sequence are well-known. See, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. 1989)and F. Eckstein (ed.) Oligonucleotides and Analogues, 1st Ed. (OxfordUniversity Press, New York, 1991). Solid-phase synthesis methods arepreferred for both oligoribonucleotides and oligodeoxyribonucleotides(the well-known methods of synthesizing DNA are also useful forsynthesizing RNA). Oligoribonucleotides and oligodeoxyribonucleotidescan also be prepared enzymatically.

[0124] The invention provides methods of detecting nucleic acids. Anytype of nucleic acid may be detected, and the methods may be used, e.g.,for the diagnosis of disease and in sequencing of nucleic acids.Examples of nucleic acids that can be detected by the methods of theinvention include genes (e.g., a gene associated with a particulardisease), viral RNA and DNA, bacterial DNA, fungal DNA, cDNA, mRNA, RNAand DNA fragments, oligonucleotides, synthetic oligonucleotides,modified oligonucleotides, single-stranded and double-stranded nucleicacids, natural and synthetic nucleic acids, etc. Thus, examples of theuses of the methods of detecting nucleic acids include: the diagnosisand/or monitoring of viral diseases (e.g., human immunodeficiency virus,hepatitis viruses, herpes viruses, cytomegalovirus, and Epstein-Barrvirus), bacterial diseases (e.g., tuberculosis, Lyme disease, H. pylori,Escherichia coli infections, Legionella infections, Mycoplasmainfections, Salmonella infections), sexually transmitted diseases (e.g.,gonorrhea), inherited disorders (e.g., cystic fibrosis, Duchene musculardystrophy, phenylketonuria, sickle cell anemia), and cancers (e.g.,genes associated with the development of cancer); in forensics; in DNAsequencing; for paternity testing; for cell line authentication; formonitoring gene therapy; and for many other purposes.

[0125] The methods of detecting nucleic acids based on observing a colorchange with the naked eye are cheap, fast, simple, robust (the reagentsare stable), do not require specialized or expensive equipment, andlittle or no instrumentation is required. This makes them particularlysuitable for use in, e.g., research and analytical laboratories in DNAsequencing, in the field to detect the presence of specific pathogens,in the doctor's office for quick identification of an infection toassist in prescribing a drug for treatment, and in homes and healthcenters for inexpensive first-line screening.

[0126] The nucleic acid to be detected may be isolated by known methods,or may be detected directly in cells, tissue samples, biological fluids(e.g., saliva, urine, blood, serum), solutions containing PCRcomponents, solutions containing large excesses of oligonucleotides orhigh molecular weight DNA, and other samples, as also known in the art.is See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual(2nd ed. 1989) and B. D. Hames and S. J. Higgins, Eds., Gene Probes 1(IRL Press, New York, 1995). Methods of preparing nucleic acids fordetection with hybridizing probes are well known in the art. See, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. 1989)and B. D. Hames and S. J. Higgins, Eds., Gene Probes 1 (IRL Press, NewYork, 1995).

[0127] If a nucleic acid is present in small amounts, it may be appliedby methods known in the art. See, e.g., Sambrook et al., MolecularCloning: A Laboratory Manual (2nd ed. 1989) and B. D. Hames and S. J.Higgins, Eds., Gene Probes 1 (IRL Press, New York, 1995). Preferred ispolymerase chain reaction (PCR) amplification.

[0128] One method according to the invention for detecting nucleic acidcomprises contacting a nucleic acid with one or more types ofnanoparticles having oligonucleotides attached thereto. The nucleic acidto be detected has at least two portions. The lengths of these portionsand the distance(s), if any, between them are chosen so that when theoligonucleotides on the nanoparticles hybridize to the nucleic acid, adetectable change occurs. These lengths and distances can be determinedempirically and will depend on the type of particle used and its sizeand the type of electrolyte which will be present in solutions used inthe assay (as is known in the art, certain electrolytes affect theconformation of nucleic acids).

[0129] Also, when a nucleic acid is to be detected in the presence ofother nucleic acids, the portions of the nucleic acid to which theoligonucleotides on the nanoparticles are to bind must be chosen so thatthey contain sufficient unique sequence so that detection of the nucleicacid will be specific. Guidelines for doing so are well known in theart.

[0130] Although nucleic acids may contain repeating sequences closeenough to each other so that only one type ofoligonucleotide-nanoparticle conjugate need be used, this will be a rareoccurrence. In general, the chosen portions of the nucleic acid willhave different sequences and will be contacted with nanoparticlescarrying two or more different oligonucleotides, preferably attached todifferent nanoparticles. An example of a system for the detection ofnucleic acid is illustrated in FIG. 2. As can be seen, a firstoligonucleotide attached to a first nanoparticle has a sequencecomplementary to a first portion of the target sequence in thesingle-stranded DNA. A second oligonucleotide attached to a secondnanoparticle has a sequence complementary to a second portion of thetarget sequence in the DNA. Additional portions of the DNA could betargeted with corresponding nanoparticles. See FIG. 17. Targetingseveral portions of a nucleic acid increases the magnitude of thedetectable change.

[0131] The contacting of the nanoparticle-oligonucleotide conjugateswith the nucleic acid takes place under conditions effective forhybridization of the oligonucleotides on the nanoparticles with thetarget sequence(s) of the nucleic acid. These hybridization conditionsare well known in the art and can readily be optimized for theparticular system employed. See, e.g., Sambrook et al., MolecularCloning: A Laboratory Manual (2nd ed. 1989). Preferably stringenthybridization conditions are employed.

[0132] Faster hybridization can be obtained by freezing and thawing asolution containing the nucleic acid to be detected and thenanoparticle-oligonucleotide conjugates. The solution may be frozen inany convenient manner, such as placing it in a dry ice-alcohol bath fora sufficient time for the solution to freeze (generally about 1 minutefor 100 μL of solution). The solution must be thawed at a temperaturebelow the thermal denaturation temperature, which can conveniently beroom temperature for most combinations of nanoparticle-oligonucleotideconjugates and nucleic acids. The hybridization is complete, and thedetectable change may be observed, after thawing the solution.

[0133] The rate of hybridization can also be increased by warming thesolution containing the nucleic acid to be detected and thenanoparticle-oligonucleotide conjugates to a temperature below thedissociation temperature (Tm) for the complex formed between theoligonucleotides on the nanoparticles and the target nucleic acid.Alternatively, rapid hybridization can be achieved by heating above thedissociation temperature (Tm) and allowing the solution to cool.

[0134] The rate of hybridization can also be increased by increasing thesalt concentration (e.g., from 0.1 M to 0.3 M NaCl).

[0135] The detectable change that occurs upon hybridization of theoligonucleotides on the nanoparticles to the nucleic acid may be a colorchange, the formation of aggregates of the nanoparticles, or theprecipitation of the aggregated nanoparticles. The color changes can beobserved with the naked eye or spectroscopically. The formation ofaggregates of the nanoparticles can be observed by electron microscopyor by nephelometry. The precipitation of the aggregated nanoparticlescan be observed with the naked eye or microscopically. Preferred arechanges observable with the naked eye. Particularly preferred is a colorchange observable with the naked eye.

[0136] The observation of a color change with the naked eye can be mademore readily against a background of a contrasting color. For instance,when gold nanoparticles are used, the observation of a color change isfacilitated by spotting a sample of the hybridization solution on asolid white surface (such as silica or alumina TLC plates, filter paper,cellulose nitrate membranes, and nylon membranes, preferably a C-18silica TLC plate) and allowing the spot to dry. Initially, the spotretains the color of the hybridization solution (which ranges frompink/red, in the absence of hybridization, to purplish-red/purple, ifthere has been hybridization). On drying at room temperature or 80° C.(temperature is not critical), a blue spot develops ifthenanoparticle-oligonucleotide conjugates had been linked by hybridizationwith the target nucleic acid prior to spotting. In the absence ofhybridization (e.g., because no target nucleic acid is present), thespot is pink. The blue and the pink spots are stable and do not changeon subsequent cooling or heating or over time. They provide a convenientpermanent record of the test. No other steps (such as a separation ofhybridized and unhybridized nanoparticle-oligonucleotide conjugates) arenecessary to observe the color change.

[0137] An alternate method for easily visualizing the assay results isto spot a sample of nanoparticle probes hybridized to a target nucleicacid on a glass fiber filter (e.g., Borosilicate Microfiber Filter, 0.7micron pore size, grade FG75, for use with gold nanoparticles 13 nm insize), while drawing the liquid through the filter. Subsequent rinsingwith water washes the excess, non-hybridized probes through the filter,leaving behind an observable spot comprising the aggregates generated byhybridization of the nanoparticle probes with the target nucleic acid(retained because these aggregates are larger than the pores of thefilter). This technique may provide for greater sensitivity, since anexcess of nanoparticle probes can be used. Unfortunately, thenanoparticle probes stick to many other solid surfaces that have beentried (silica slides, reverse-phase plates, and nylon, nitrocellulose,cellulose and other membranes), and these surfaces cannot be used.

[0138] An important aspect of the detection system illustrated in FIG. 2is that obtaining a detectable change depends on cooperativehybridization of two different oligonucleotides to a given targetsequence in the nucleic acid. Mismatches in either of the twooligonucleotides will destabilize the interparticle connection. It iswell known that a mismatch in base pairing has a much greaterdestabilizing effect on the binding of a short oligonucleotide probethan on the binding of a long oligonucleotide probe. The advantage ofthe system illustrated in FIG. 2 is that it utilizes the basediscrimination associated with a long target sequence and probe(eighteen base-pairs in the example illustrated in FIG. 2), yet has thesensitivity characteristic of a short oligonucleotide probe (ninebase-pairs in the example illustrated in FIG. 2).

[0139] The target sequence of the nucleic acid may be contiguous, as inFIG. 2, or the two portions of the target sequence may be separated by athird portion which is not complementary to the oligonucleotides on thenanoparticles, as illustrated in FIG. 3. In the latter case, one has theoption of using a filler oligonucleotide which is free in solution andwhich has a sequence complementary to that of this third portion (seeFIG. 3). When the filler oligonucleotide hybridizes with the thirdportion of the nucleic acid, a double-stranded segment is created,thereby altering the average distance between the nanoparticles and,consequently, the color. The system illustrated in FIG. 3 may increasethe sensitivity of the detection method.

[0140] Some embodiments of the method of detecting nucleic acid utilizea substrate. By employing a substrate, the detectable change (thesignal) can be amplified and the sensitivity of the assay increased.

[0141] Any substrate can be used which allows observation of thedetectable change. Suitable substrates include transparent solidsurfaces (e.g., glass, quartz, plastics and other polymers), opaquesolid surface (e.g., white solid surfaces, such as TLC silica plates,filter paper, glass fiber filters, cellulose nitrate membranes, nylonmembranes), and conducting solid surfaces (e.g., indium-tin-oxide(ITO)). The substrate can be any shape or thickness, but generally willbe flat and thin. Preferred are transparent substrates such as glass(e.g., glass slides) or plastics (e.g., wells of microtiter plates).

[0142] In one embodiment, oligonucleotides are attached to thesubstrate. The oligonucleotides can be attached to the substrates asdescribed in, e.g., Chrisey et al., Nucleic Acids Res., 24, 3031-3039(1996); Chrisey et al., Nucleic Acids Res., 24, 3040-3047 (1996); Mucicet al., Chem. Commun., 555 (1996); Zimmermann and Cox, Nucleic AcidsRes., 22, 492 (1994); Bottomley et al., J. Vac. Sci. Technol. A, 10, 591(1992); and Hegner et al., FEBS Lett., 336, 452 (1993).

[0143] The oligonucleotides attached to the substrate have a sequencecomplementary to a first portion of the sequence of a nucleic acid to bedetected. The nucleic acid is contacted with the substrate underconditions effective to allow hybridization of the oligonucleotides onthe substrate with the nucleic acid. In this manner the nucleic acidbecomes bound to the substrate. Any unbound nucleic acid is preferablywashed from the substrate before adding nanoparticle-oligonucleotideconjugates.

[0144] Next, the nucleic acid bound to the substrate is contacted with afirst type of nanoparticles having oligonucleotides attached thereto.The oligonucleotides have a sequence complementary to a second portionof the sequence of the nucleic acid, and the contacting takes placeunder conditions effective to allow hybridization of theoligonucleotides on the nanoparticles with the nucleic acid. In thismanner the first type of nanoparticles become bound to the substrate.After the nanoparticle-oligonucleotide conjugates are bound to thesubstrate, the substrate is washed to remove any unboundnanoparticle-oligonucleotide conjugates and nucleic acid.

[0145] The oligonucleotides on the first type of nanoparticles may allhave the same sequence or may have different sequences that hybridizewith different portions of the nucleic acid to be detected. Whenoligonucleotides having different sequences are used, each nanoparticlemay have all of the different oligonucleotides attached to it or,preferably, the different oligonucleotides are attached to differentnanoparticles. FIG. 17 illustrates the use ofnanoparticle-oligonucleotide conjugates designed to hybridize tomultiple portions of a nucleic acid. Alternatively, the oligonucleotideson each of the first type of nanoparticles may have a plurality ofdifferent sequences, at least one of which must hybridize with a portionof the nucleic acid to be detected (see FIG. 25B).

[0146] Finally, the first type of nanoparticle-oligonucleotideconjugates bound to the substrate is contacted with a second type ofnanoparticles having oligonucleotides attached thereto. Theseoligonucleotides have a sequence complementary to at least a portion ofthe sequence(s) of the oligonucleotides attached to the first type ofnanoparticles, and the contacting takes place under conditions effectiveto allow hybridization of the oligonucleotides on the first type ofnanoparticles with those on the second type of nanoparticles. After thenanoparticles are bound, the substrate is preferably washed to removeany unbound nanoparticle-oligonucleotide conjugates.

[0147] The combination of hybridizations produces a detectable change.The detectable changes are the same as those described above, exceptthat the multiple hybridizations result in an amplification of thedetectable change. In particular, since each of the first type ofnanoparticles has multiple oligonucleotides (having the same ordifferent sequences) attached to it, each of the first type ofnanoparticle-oligonucleotide conjugates can hybridize to a plurality ofthe second type of nanoparticle-oligonucleotide conjugates. Also, thefirst type of nanoparticle-oligonucleotide conjugates may be hybridizedto more than one portion of the nucleic acid to be detected. Theamplification provided by the multiple hybridizations may make thechange detectable for the first time or may increase the magnitude ofthe detectable change. This amplification increases the sensitivity ofthe assay, allowing for detection of small amounts of nucleic acid.

[0148] If desired, additional layers of nanoparticles can be built up bysuccessive additions of the first and second types ofnanoparticle-oligonucleotide conjugates. In this way, the number ofnanoparticles immobilized per molecule of target nucleic acid can befurther increased with a corresponding increase in intensity of thesignal.

[0149] Also, instead of using first and second types ofnanoparticle-oligonucleotide conjugates designed to hybridize to eachother directly, nanoparticles bearing oligonucleotides that would serveto bind the nanoparticles together as a consequence of hybridizationwith binding oligonucleotides could be used.

[0150] Methods of making the nanoparticles and the oligonucleotides andof attaching the oligonucleotides to the nanoparticles are describedabove. The hybridization conditions are well known in the art and can bereadily optimized for the particular system employed (see above).

[0151] An example of this method of detecting nucleic acid (analyte DNA)is illustrated in FIG. 13A. As shown in that Figure, the combination ofhybridizations produces dark areas where nanoparticle aggregates arelinked to the substrate by analyte DNA. These dark areas may be readilyobserved with the naked eye using ambient light, preferably viewing thesubstrate against a white background. As can be readily seen from FIG.13A, this method provides a means of amplifying a detectable change.

[0152] Another example of this method of detecting nucleic acid isillustrated in FIG. 25B. As in the example illustrated in FIG. 13A, thecombination of hybridizations produces dark areas where nanoparticleaggregates are linked to the substrate by analyte DNA which can beobserved with the naked eye.

[0153] In another embodiment, nanoparticles are attached to thesubstrate. Nanoparticles can be attached to substrates as described in,e.g., Grabar et al., Analyt. Chem., 67, 73-743 (1995); Bethell et al.,J. Electroanal. Chem., 409, 137 (1996); Baretal., Langmuir, 12,1172(1996); Colvin et al., J. Am. Chem. Soc., 114, 5221 (1992).

[0154] After the nanoparticles are attached to the substrate,oligonucleotides are attached to the nanoparticles. This may beaccomplished in the same manner described above for the attachment ofoligonucleotides to nanoparticles in solution. The oligonucleotidesattached to the nanoparticles have a sequence complementary to a firstportion of the sequence of a nucleic acid.

[0155] The substrate is contacted with the nucleic acid under conditionseffective to allow hybridization of the oligonucleotides on thenanoparticles with the nucleic acid. In this manner the nucleic acidbecomes bound to the substrate. Unbound nucleic acid is preferablywashed from the substrate prior to adding furthernanoparticle-oligonucleotide conjugates.

[0156] Then, a second type of nanoparticles having oligonucleotidesattached thereto is provided. These oligonucleotides have a sequencecomplementary to a second portion of the sequence of the nucleic acid,and the nucleic acid bound to the substrate is contacted with the secondtype of nanoparticle-oligonucleotide conjugates under conditionseffective to allow hybridization of the oligonucleotides on the secondtype of nanoparticle-oligonucleotide conjugates with the nucleic acid.In this manner, the second type of nanoparticle-oligonucleotideconjugates becomes bound to the substrate. After the nanoparticles arebound, any unbound nanoparticle-oligonucleotide conjugates and nucleicacid are washed from the substrate. A change (e.g., color change) may bedetectable at this point.

[0157] The oligonucleotides on the second type of nanoparticles may allhave the same sequence or may have different sequences that hybridizewith different portions of the nucleic acid to be detected. Whenoligonucleotides having different sequences are used, each nanoparticlemay have all of the different oligonucleotides attached to it or,preferably, the different oligonucleotides may be attached to differentnanoparticles. See FIG. 17.

[0158] Next, a binding oligonucleotide having a selected sequence havingat least two portions, the first portion being complementary to at leasta portion of the sequence of the oligonucleotides on the second type ofnanoparticles, is contacted with the second type ofnanoparticle-oligonucleotide conjugates bound to the substrate underconditions effective to allow hybridization of the bindingoligonucleotide to the oligonucleotides on the nanoparticles. In thismanner, the binding oligonucleotide becomes bound to the substrate.After the binding oligonucleotides are bound, unbound bindingoligonucleotides are washed from the substrate.

[0159] Finally, a third type of nanoparticles having oligonucleotidesattached thereto is provided. The oligonucleotides have a sequencecomplementary to the sequence of a second portion of the bindingoligonucleotide. The nanoparticle-oligonucleotide conjugates arecontacted with the binding oligonucleotide bound to the substrate underconditions effective to allow hybridization of the bindingoligonucleotide to the oligonucleotides on the nanoparticles. After thenanoparticles are bound, unbound nanoparticle-oligonucleotide conjugatesare washed from the substrate.

[0160] The combination of hybridizations produces a detectable change.The detectable changes are the same as those described above, exceptthat the multiple hybridizations result in an amplification of thedetectable change. In particular, since each of the second type ofnanoparticles has multiple oligonucleotides (having the same ordifferent sequences) attached to it, each of the second type ofnanoparticle-oligonucleotide conjugates can hybridize to a plurality ofthe third type of nanoparticle-oligonucleotide conjugates (through thebinding oligonucleotide). Also, the second type ofnanoparticle-oligonucleotide conjugates may be hybridized to more thanone portion of the nucleic acid to be detected. The amplificationprovided by the multiple hybridizations may make the change detectablefor the first time or may increase the magnitude of the detectablechange. The amplification increases the sensitivity of the assay,allowing for detection of small amounts of nucleic acid.

[0161] If desired, additional layers of nanoparticles can be built up bysuccessive additions of the binding oligonucleotides and second andthird types of nanoparticle-oligonucleotide conjugates. In this way, thenanoparticles immobilized per molecule of target nucleic acid can befurther increased with a corresponding increase in intensity of thesignal.

[0162] Also, the use of the binding oligonucleotide can be eliminated,and the second and third types of nanoparticle-oligonucleotideconjugates can be designed so that they hybridize directly to eachother.

[0163] Methods of making the nanoparticles and the oligonucleotides andof attaching the oligonucleotides to the nanoparticles are describedabove. The hybridization conditions are well known in the art and can bereadily optimized for the particular system employed (see above).

[0164] An example of this method of detecting nucleic acid (analyte DNA)is illustrated in FIG. 13B. As shown in that Figure, the combination ofhybridizations produces dark areas where nanoparticle aggregates arelinked to the substrate by analyte DNA. These dark areas may be readilyobserved with the naked eye as described above. As can be seen from FIG.13B, this embodiment of the method of the invention provides anothermeans of amplifying the detectable change.

[0165] Another amplification scheme employs liposomes. In this scheme,oligonucleotides are attached to a substrate. Suitable substrates arethose described above, and the oligonucleotides can be attached to thesubstrates as described above. For instance, where the substrate isglass, this can be accomplished by condensing the oligonucleotidesthrough phosphoryl or carboxylic acid groups to aminoalkyl groups on thesubstrate surface (for related chemistry see Grabar et al., Anal. Chem.,67, 735-743 (1995)).

[0166] The oligonucleotides attached to the substrate have a sequencecomplementary to a first portion of the sequence of the nucleic acid tobe detected. The nucleic acid is contacted with the substrate underconditions effective to allow hybridization of the oligonucleotides onthe substrate with the nucleic acid. In this manner the nucleic acidbecomes bound to the substrate. Any unbound nucleic acid is preferablywashed from the substrate before adding additional components of thesystem.

[0167] Next, the nucleic acid bound to the substrate is contacted withliposomes having oligonucleotides attached thereto. The oligonucleotideshave a sequence complementary to a second portion of the sequence of thenucleic acid, and the contacting takes place under conditions effectiveto allow hybridization of the oligonucleotides on the liposomes with thenucleic acid. In this manner the liposomes become bound to thesubstrate. After the liposomes are bound to the substrate, the substrateis washed to remove any unbound liposomes and nucleic acid.

[0168] The oligonucleotides on the liposomes may all have the samesequence or may have different sequences that hybridize with differentportions of the nucleic acid to be detected. When oligonucleotideshaving different sequences are used, each liposome may have all of thedifferent oligonucleotides attached to it or the differentoligonucleotides may be attached to different liposomes.

[0169] To prepare oligonucleotide-liposome conjugates, theoligonucleotides are linked to a hydrophobic group, such as cholesteryl(see Letsinger et al., J. Am. Chem. Soc., 115,7535-7536 (1993)), and thehydrophobic-oligonucleotide conjugates are mixed with a solution ofliposomes to form liposomes with hydrophobic-oligonucleotide conjugatesanchored in the membrane (see Zhang et al., Tetrahedron Lett., 37,6243-6246 (1996)). The loading of hydrophobic-oligonucleotide conjugateson the surface of the liposomes can be controlled by controlling theratio of hydrophobic-oligonucleotide conjugates to liposomes in themixture. It has been observed that liposomes bearing oligonucleotidesattached by hydrophobic interaction of pendent cholesteryl groups areeffective in targeting polynucleotides immobilized on a nitrocellulosemembrane (Id.). Fluorescein groups anchored in the membrane of theliposome were used as the reporter group. They served effectively, butsensitivity was limited by the fact that the signal from fluorescein inregions of high local concentration (e.g., on the liposome surface) isweakened by self quenching.

[0170] The liposomes are made by methods well known in the art. SeeZhang et al., Tetrahedron Lett., 37, 6243 (1996). The liposomes willgenerally be about 5-50 times larger in size (diameter) than thenanoparticles used in subsequent steps. For instance, for nanoparticlesabout 13 nm in diameter, liposomes about 100 rn in diameter arepreferably used.

[0171] The liposomes bound to the substrate are contacted with a firsttype of nanoparticles having at least a first type of oligonucleotidesattached thereto. The first type of oligonucleotides have a hydrophobicgroup attached to the end not attached to the nanoparticles, and thecontacting takes place under conditions effective to allow attachment ofthe oligonucleotides on the nanoparticles to the liposomes as a resultof hydrophobic interactions. A detectable change may be observable atthis point.

[0172] The method may further comprise contacting the first type ofnanoparticle-oligonucleotide conjugates bound to the liposomes with asecond type of nanoparticles having oligonucleotides attached thereto.The first type of nanoparticles have a second type of oligonucleotidesattached thereto which have a sequence complementary to at least aportion of the sequence of the oligonucleotides on the second type ofnanoparticles, and the oligonucleotides on the second type ofnanoparticles have a sequence complementary to at least a portion of thesequence of the second type of oligonucleotides on the first type ofnanoparticles. The contacting takes place under conditions effective toallow hybridization of the oligonucleotides on the first and secondtypes of nanoparticles. This hybridization will generally be performedat mild temperatures (e.g., 5° C. to 60° C.), so conditions (e.g.,0.3-1.0 M NaCl) conducive to hybridization at room temperature areemployed. Following hybridization, unbound nanoparticle-oligonucleotideconjugates are washed from the substrate.

[0173] The combination of hybridizations produces a detectable change.The detectable changes are the same as those described above, exceptthat the multiple hybridizations result in an amplification of thedetectable change. In particular, since each of the liposomes hasmultiple oligonucleotides (having the same or different sequences)attached to it, each of the liposomes can hybridize to a plurality ofthe first type of nanoparticle-oligonucleotide conjugates. Similarly,since each of the first type of nanoparticles has multipleoligonucleotides attached to it, each of the first type ofnanoparticle-oligonucleotide conjugates can hybridize to a plurality ofthe second type of nanoparticle-oligonucleotide conjugates. Also, theliposomes may be hybridized to more than one portion of the nucleic acidto be detected. The amplification provided by the multiplehybridizations may make the change detectable for the first time or mayincrease the magnitude of the detectable change. This amplificationincreases the sensitivity of the assay, allowing for detection of smallamounts of nucleic acid.

[0174] If desired, additional layers of nanoparticles can be built up bysuccessive additions of the first and second types ofnanoparticle-oligonucleotide conjugates. In this way, the number ofnanoparticles immobilized per molecule of target nucleic acid can befurther increased with a corresponding increase in the intensity of thesignal.

[0175] Also, instead of using second and third types ofnanoparticle-oligonucleotide conjugates designed to hybridize to eachother directly, nanoparticles bearing oligonucleotides that would serveto bring the nanoparticles together as a consequence of hybridizationwith binding oligonucleotides could be used.

[0176] Methods of making the nanoparticles and the oligonucleotides andof attaching the oligonucleotides to the nanoparticles are describedabove. A mixture of oligonucleotides functionalized at one end forbinding to the nanoparticles and with or without a hydrophobic group atthe other end can be used on the first type of nanoparticles. Therelative ratio of these oligonucleotides bound to the averagenanoparticle will be controlled by the ratio of the concentrations ofthe two oligonucleotides in the mixture. The hybridization conditionsare well known in the art and can be readily optimized for theparticular system employed (see above).

[0177] An example of this method of detecting nucleic acid isillustrated in FIG. 18. The hybridization of the first type ofnanoparticle-oligonucleotide conjugates to the liposomes may produce adetectable change. In the case of gold nanoparticles, a pink/red colormay be observed or a purple/blue color may be observed if thenanoparticles are close enough together. The hybridization of the secondtype of nanoparticle-oligonucleotide conjugates to the first type ofnanoparticle-oligonucleotide conjugates will produce a detectablechange. In the case of gold nanoparticles, a purple/blue color will beobserved. All of these color changes may be observed with the naked eye.

[0178] In yet other embodiments utilizing a substrate, an “aggregateprobe” can be used. The aggregate probe can be prepared by allowing twotypes of nanoparticles having complementary oligonucleotides (a and a′)attached to them to hybridize to form a core (illustrated in FIG. 28A).Since each type of nanoparticle has a plurality of oligonucleotidesattached to it, each type of nanoparticles will hybridize to a pluralityof the other type of nanoparticles. Thus, the core is an aggregatecontaining numerous nanoparticles of both types. The core is then cappedwith a third type of nanoparticles having at least two types ofoligonucleotides attached to them. The first type of oligonucleotideshas a sequence b which is complementary to the sequence b′ of a portionof a nucleic acid to be detected. The second type of oligonucleotideshas sequence a or a′ so that the third type of nanoparticles willhybridize to nanoparticles on the exterior of the core. The aggregateprobe can also be prepared by utilizing two types of nanoparticles (seeFIG. 28B). Each type of nanoparticles has at least two types ofoligonucleotides attached to them. The first type of oligonucleotidespresent on each of the two types of nanoparticles has sequence b whichis complementary to the sequence b′ of a portion of the nucleic acid tobe detected. The second type of oligonucleotides on the first type ofnanoparticles has a sequence a which is complementary to the sequence a′of the second type of oligonucleotides on the second type ofnanoparticles (see FIG. 28B) so that the two types of nanoparticleshybridize to each other to form the aggregate probe. Since each type ofnanoparticles has a plurality of oligonucleotides attached to it, eachtype of nanoparticles will hybridize to a plurality of the other type ofnanoparticles to form an aggregate containing numerous nanoparticles ofboth types.

[0179] The aggregate probe can be utilized to detect nucleic acid in anyof the above assay formats performed on a substrate, eliminating theneed to build up layers of individual nanoparticles in order to obtainor enhance a detectable change. To even further enhance the detectablechange, layers of aggregate probes can be built up by using two types ofaggregate probes, the first type of aggregate probe havingoligonucleotides attached to it that are complementary tooligonucleotides on the other type of aggregate probe. In particular,when the aggregate probe is prepared as illustrated in FIG. 28B, theaggregate probes can hybridize to each other to form the multiplelayers. Some of the possible assay formats utilizing aggregate probesare illustrated in FIGS. 28C-D. For instance, a type of oligonucleotidescomprising sequence c is attached to a substrate (see FIG. 28C).Sequence c is complementary to the sequence c′ of a portion of a nucleicacid to be detected. The target nucleic acid is added and allowed tohybridize to the oligonucleotides attached to the substrate, after whichthe aggregate probe is added and allowed to hybridize to the portion ofthe target nucleic acid having sequence b′, thereby producing adetectable change. Alternatively, the target nucleic acid can first behybridized to the aggregate probe in solution and subsequentlyhybridized to the oligonucleotides on the substrate, or the targetnucleic acid can simultaneously be hybridized to the aggregate probe andthe oligonucleotides on the substrate. In another embodiment, the targetnucleic acid is allowed to react with the aggregate probe and anothertype of nanoparticles in solution (see FIG. 28D). Some of theoligonucleotides attached to this additional type of nanoparticlescomprise sequence c so that they hybridize to sequence c′ of the targetnucleic acid and some of the oligonucleotides attached to thisadditional type of nanoparticles comprise sequence d so that they cansubsequently hybridize to oligonucleotides comprising sequence d′ whichare attached to the substrate.

[0180] The core itself can also be used as a probe to detect nucleicacids. One possible assay format is illustrated in FIG. 28E. Asillustrated there, a type of oligonucleotides comprising sequence b isattached to a substrate. Sequence b is complementary to the sequence b′of a portion of a nucleic acid to be detected. The target nucleic acidis contacted with the substrate and allowed to hybridize to theoligonucleotides attached to the substrate. Then, another type ofnanoparticles is added. Some of the oligonucleotides attached to thisadditional type of nanoparticles comprise sequence c so which iscomplementary to sequence c′ of the target nucleic acid so that thenanoparticles hybridize to the target nucleic acid bound to thesubstrate. Some of the oligonucleotides attached to the additional typeof nanoparticles comprise sequence a or a′ complementary to sequences aand a′ on the core probe, and the core probe is added and allowed tohybridize to the oligonucleotides on the nanoparticles. Since each coreprobe has sequences a and a′ attached to the nanoparticles whichcomprise the core, the core probes can hybridize to each other to formmultiple layers attached to the substrate, providing a greatly enhanceddetectable change. In alternative embodiments, the target nucleic acidcould be contacted with the additional type of nanoparticles in solutionprior to being contacted with the substrate, or the target nucleic acid,the nanoparticles and the substrate could all be contactedsimultaneously. In yet another alternative embodiment, the additionaltype of nanoparticles could be replaced by a linking oligonucleotidecomprising both sequences c and a or a′.

[0181] When a substrate is employed, a plurality of the initial types ofnanoparticle-oligonucleotide conjugates or oligonucleotides can beattached to the substrate in an array for detecting multiple portions ofa target nucleic acid, for detecting multiple different nucleic acids,or both. For instance, a substrate may be provided with rows of spots,each spot containing a different type of oligonucleotide oroligonucleotide-nanoparticle conjugate designed to bind to a portion ofa target nucleic acid. A sample containing one or more nucleic acids isapplied to each spot, and the rest of the assay is performed in one ofthe ways described above using appropriate oligonucleotide-nanoparticleconjugates, oligonucleotide-liposome conjugates, aggregate probes, coreprobes, and binding oligonucleotides.

[0182] Finally, when a substrate is employed, a detectable change can beproduced or further enhanced by silver staining. Silver staining can beemployed with any type of nanoparticles that catalyze the reduction ofsilver. Preferred are nanoparticles made of noble metals (e.g., gold andsilver). See Bassell, et al., J. Cell Biol., 126, 863-876 (1994);Braun-Howland et al., Biotechniques, 13, 928-931 (1992). If thenanoparticles being employed for the detection of a nucleic acid do notcatalyze the reduction of silver, then silver ions can be complexed tothe nucleic acid to catalyze the reduction. See Braun et al., Nature,391, 775 (1998). Also, silver stains are known which can react with thephosphate groups on nucleic acids.

[0183] Silver staining can be used to produce or enhance a detectablechange in any assay performed on a substrate, including those describedabove. In particular, silver staining has been found to provide a hugeincrease in sensitivity for assays employing a single type ofnanoparticle, such as the one illustrated in FIG. 25A, so that the useof layers of nanoparticles, aggregate probes and core probes can oftenbe eliminated.

[0184] In assays for detecting nucleic acids performed on a substrate,the detectable change can be observed with an optical scanner. Suitablescanners include those used to scan documents into a computer which arecapable of operating in the reflective mode (e.g., a flatbed scanner),other devices capable of performing this function or which utilize thesame type of optics, any type of greyscale-sensitive measurement device,and standard scanners which have been modified to scan substratesaccording to the invention (e.g., a flatbed scanner modified to includea holder for the substrate) (to date, it has not been found possible touse scanners operating in the transmissive mode). The resolution of thescanner must be sufficient so that the reaction area on the substrate islarger than a single pixel of the scanner. The scanner can be used withany substrate, provided that the detectable change produced by the assaycan be observed against the substrate (e.g., a grey spot, such as thatproduced by silver staining, can be observed against a white background,but cannot be observed against a grey background). The scanner can be ablack-and-white scanner or, preferably, a color scanner. Mostpreferably, the scanner is a standard color scanner of the type used toscan documents into computers. Such scanners are inexpensive and readilyavailable commercially. For instance, an Epson Expression 636 (600×600dpi), a UMAX Astra 1200 (300×300 dpi), or a Microtec 1600 (1600×1600dpi) can be used. The scanner is linked to a computer loaded withsoftware for processing the images obtained by scanning the substrate.The software can be standard software which is readily availablecommercially, such as Adobe Photoshop 5.2 and Corel Photopaint 8.0.Using the software to calculate greyscale measurements provides a meansof quantitating the results of the assays. The software can also providea color number for colored spots and can generate images (e.g.,printouts) of the scans which can be reviewed to provide a qualitativedetermination of the presence of a nucleic acid, the quantity of anucleic acid, or both. In addition, it has been found that thesensitivity of assays such as that described in Example 5 can beincreased by subtracting the color that represents a negative result(red in Example 5) from the color that represents a positive result(blue in Example 5). The computer can be a standard personal computerwhich is readily available commercially. Thus, the use of a standardscanner l inked to a standard computer loaded with standard software canprovide a convenient, easy, inexpensive means of detecting andquantitating nucleic acids when the assays are performed on substrates.The scans can also be stored in the computer to maintain a record of theresults for further reference or use. Of course, more sophisticatedinstruments and software can be used, if desired.

[0185] A nanoparticle-oligonucleotide conjugate which may be used in anassay for any nucleic acid is illustrated in FIGS. 17D-E. This“universal probe” has oligonucleotides of a single sequence attached toit. These oligonucleotides can hybridize with a binding oligonucleotidewhich has a sequence comprising at least two portions. The first portionis complementary to at least a portion of the sequence of theoligonucleotides on the nanoparticles. The second portion iscomplementary to a portion of the sequence of the nucleic acid to bedetected. A plurality of binding oligonucleotides having the same firstportion and different second portions can be used, in which case the“universal probe”, after hybridization to the binding oligonucleotides,can bind to multiple portions of the nucleic acid to be detected or todifferent nucleic acid targets.

[0186] In a number of other embodiments of the invention, the detectablechange is created by labeling the oligonucleotides, the nanoparticles,or both with molecules (e.g., fluorescent molecules and dyes) thatproduce detectable changes upon hydridization of the oligonucleotides onthe nanoparticles with the target nucleic acid. For instance,oligonucleotides attached to metal and semiconductor nanoparticles canhave a fluorescent molecule attached to the end not attached to thenanoparticles. Metal and semiconductor nanoparticles are knownfluorescence quenchers, with the magnitude of the quenching effectdepending on the distance between the nanoparticles and the fluorescentmolecule. In the unhybridized state, the oligonucleotides attached tothe nanoparticles interact with the nanoparticles, so that significantquenching will be observed. See FIG. 20A. Upon hybridization to a targetnucleic acid, the fluorescent molecule will become spaced away from thenanoparticles, diminishing quenching of the fluorescence. See FIG. 20A.Longer oligonucleotides should give rise to larger changes influorescence, at least until the fluorescent groups are moved far enoughaway from the nanoparticle surfaces so that an increase in the change isno longer observed. Useful lengths of the oligonucleotides can bedetermined empirically. Metallic and semiconductor nanoparticles havingfluorescent-labeled oligonucleotides attached thereto can be used in anyof the assay formats described above, including those performed insolution or on substrates.

[0187] Methods of labeling oligonucleotides with fluorescent moleculesand measuring fluorescence are well known in the art. Suitablefluorescent molecules are also well known in the art and include thefluoresceins, rhodamines and Texas Red. The oligonucleotides will beattached to the nanoparticles as described above.

[0188] In yet another embodiment, two types of fluorescent-labeledoligonucleotides attached to two different particles can be used.Suitable particles include polymeric particles (such as polystyreneparticles, polyvinyl particles, acrylate and methacrylate particles),glass particles, latex particles, Sepharose beads and others likeparticles well known in the art. Methods of attaching oligonucleotidesto such particles are well known in the art. See Chrisey et al., NucleicAcids Research, 24, 3031-3039 (1996) (glass) and Charreyre et al.,Langmuir, 13,3103-3110 (1997), Fahy et al., Nucleic Acids Research,21,1819-1826 (1993), Elaissari et al., J. Colloid Interface Sci., 202,251-260 (1998), Kolarova et al., Biotechniques, 20, 196-198 (1996) andWolf et al., Nucleic Acids Research, 15, 2911-2926 (1987)(polymer/latex). In particular, a wide variety of functional groups areavailable on the particles or can be incorporated into such particles.Functional groups include carboxylic acids, aldehydes, amino groups,cyano groups, ethylene groups, hydroxyl groups, mercapto groups, and thelike. Nanoparticles, including metallic and semiconductor nanoparticles,can also be used.

[0189] The two fluorophores are designated d and a for donor andacceptor. A variety of fluorescent molecules useful in such combinationsare well known in the art and are available from, e.g., MolecularProbes. An attractive combination is fluorescein as the donor and TexasRed as acceptor. The two types of nanoparticle-oligonucleotideconjugates with d and a attached are mixed with the target nucleic acid,and fluorescence measured in a fluorimeter. The mixture will be excitedwith light of the wavelength that excites d, and the mixture will bemonitored for fluorescence from a. Upon hybridization, d and a will bebrought in proximity (see FIG. 20B). In the case of non-metallic,non-semiconductor particles, hybridization will be shown by a shift influorescence from that for d to that for a or by the appearance offluorescence for a in addition to that for d. In the absence ofhybridization, the flurophores will be too far apart for energy transferto be significant, and only the fluorescence of d will be observed. Inthe case of metallic and semiconductor nanoparticles, lack ofhybridization will be shown by a lack of fluorescence due to d or abecause of quenching (see above). Hybridization will be shown by anincrease in fluorescence due to a.

[0190] As will be appreciated, the above described particles andnanoparticles having oligonucleotides labeled with acceptor and donorfluorescent molecules attached can be used in the assay formatsdescribed above, including those performed in solution and onsubstrates. For solution formats, the oligonucleotide sequences arepreferably chosen so that they bind to the target nucleic acid asillustrated in FIGS. 15A-G. In the formats shown in FIG. 13A-B and 18,the binding oligonucleotides may be used to bring the acceptor and donorfluorescent molecules on the two nanoparticles in proximity. Also, inthe format illustrated in FIG. 13A, the oligonucleotides attached thesubstrate may be labeled with d. Further, other labels besidesfluorescent molecules can be used, such as chemiluminescent molecules,which will give a detectable signal or a change in detectable signalupon hybridization.

[0191] Another embodiment of the detection method of the invention is avery sensitive system that utilizes detection of changes in fluorescenceand color (illustrated in FIG. 21). This system employs latexmicrospheres to which are attached oligonucleotides labeled with afluorescent molecule and gold nanoparticles to which are attachedoligonucleotides. The oligonucleotide-nanoparticle conjugates can beprepared as described above. Methods of attaching oligonucleotides tolatex microspheres are well known (see, e.g., Charreyre et al.,Langmuir, 13:3103-3110 (1997); Elaissari et al., J. Colloid InterfaceSci., 202:251-260 (1998)), as are methods of labeling oligonucleotideswith fluorescent molecules (see above). The oligonucleotides on thelatex microspheres and the oligonucleotides on the gold nanoparticleshave sequences capable of hybridizing with different portions of thesequence of a target nucleic acid, but not with each other. When atarget nucleic acid comprising sequences complementary to the sequencesof the oligonucleotides on the latex microspheres and gold nanoparticlesis contacted with the two probes, a network structure is formed (seeFIG. 21). Due to the quenching properties of the gold nanoparticles, thefluorescence of the oligonucleotides attached to the latex microspheresis quenched while part of this network. Indeed, one gold nanoparticlecan quench many fluorophore molecules since gold nanoparticles have verylarge absorption coefficients. Thus, the fluorescence of a solutioncontaining nucleic acid and the two particles can be monitored to detectthe results, with a reduction in, or elimination of, fluorescenceindicating a positive result. Preferably, however, the results of theassay are detected by placing a droplet of the solution onto amicroporous material (see FIG. 21). The microporous material should betransparent or a color (e.g., white) which allows for detection of thepink/red color of the gold nanoparticles. The microporous materialshould also have a pore size sufficiently large to allow the goldnanoparticles to pass through the pores and sufficiently small to retainthe latex microspheres on the surface of the microporous material whenthe microporous material is washed. Thus, when using such a microporousmaterial, the size (diameter) of the latex microspheres must be largerthan the size (diameter) of the gold nanoparticles. The microporousmaterial must also be inert to biological media. Many suitablemicroporous materials are known in the art and include various filtersand membranes, such as modified polyvinylidene fluoride (PVDF, such asDurapore™ membrane filters purchased from Millipore Corp.) and purecellulose acetate (such as AcetatePlus™ membrane filters purchased fromMicron Separations Inc.). Such a microporous material retains thenetwork composed oftarget nucleic acid and the two probes, and apositive result (presence of the target nucleic acid) is evidenced by ared/pink color (due to the presence of the gold nanoparticles) and alack of fluorescence (due to quenching of fluorescence by the goldnanoparticles) (see FIG. 21). A negative result (no target nucleic acidpresent) is evidenced by a white color and fluorescence, because thegold nanoparticles would pass through the pores of the microporousmaterial when it is washed (so no quenching of the fluorescence wouldoccur), and the white latex microspheres would be trapped on top of it(see FIG. 21). In addition, in the case of a positive result, changes influorescence and color can be observed as a function of temperature. Forinstance, as the temperature is raised, fluorescence will be observedonce the dehybridization temperature has been reached. Therefore, bylooking at color or fluorescence as a function of temperature,information can be obtained about the degree of complementarity betweenthe oligonucleotide probes and the target nucleic acid. As noted above,this detection method exhibits high sensitivity. As little as 3femtomoles of single-stranded target nucleic acid 24 bases in length and20 femtomoles of double-stranded target nucleic acid 24 bases in lengthhave been detected with the naked eye. The method is also very simple touse. Fluorescence can be generated by simply illuminating the solutionor microporous material with a UV lamp, and the fluorescent andcolorimetric signals can be monitored by the naked eye. Alternatively,for a more quantitative result, a fluorimeter can be employed infront-face mode to measure the fluorescence of the solution with a shortpathlength.

[0192] The above embodiment has been described with particular referenceto latex microspheres and gold nanoparticles. Any other microsphere ornanoparticle, having the other properties described above and to whicholigonucleotides can be attached, can be used in place of theseparticles. Many suitable particles and nanoparticles are describedabove, along with techniques for attaching oligonucleotides to them. Inaddition, microspheres and nanoparticles having other measurableproperties may be used. For instance, polymer-modified particles andnanoparticles, where the polymer can be modified to have any desirableproperty, such as fluorescence, color, or electrochemical activity, canbe used. See, Watson et al., J. Am. Chem. Soc., 121, 462-463 (1999)(polymer-modified gold nanoparticles). Also, magnetic, polymer-coatedmagnetic, and semiconducting particles can be used. See Chan et al.,Science, 281, 2016 (1998); Bruchez et al., Science, 281, 2013 (1998);Kolarova et al., Biotechniques, 20, 196-198 (1996).

[0193] In yet another embodiment, two probes comprising metallic orsemiconductor nanoparticles having oligonucleotides labeled withfluorescent molecules attached to them are employed (illustrated in FIG.22). The oligonucleotide-nanoparticle conjugates can be prepared andlabeled with fluorescent molecules as described above. Theoligonucleotides on the two types of oligonucleotide-nanoparticleconjugates have sequences capable of hybridizing with different portionsof the sequence of a target nucleic acid, but not with each other. Whena target nucleic acid comprising sequences complementary to thesequences of the oligonucleotides on the nanoparticles is contacted withthe two probes, a network structure is formed (see FIG. 22). Due to thequenching properties of the metallic or semiconductor nanoparticles, thefluorescence of the oligonucleotides attached to the nanoparticles isquenched while part of this network. Thus, the fluorescence of asolution containing nucleic acid and the two probes can be monitored todetect the results, with a reduction in, or elimination of, fluorescenceindicating a positive result. Preferably, however, the results of theassay are detected by placing a droplet of the solution onto amicroporous material (see FIG. 22). The microporous material should havea pore size sufficiently large to allow the nanoparticles to passthrough the pores and sufficiently small to retain the network on thesurface of the microporous material when the microporous material iswashed (see FIG. 22). Many suitable microporous materials are known inthe art and include those described above. Such a microporous materialretains the network composed of target nucleic acid and the two probes,and a positive result (presence of the target nucleic acid) is evidencedby a lack of fluorescence (due to quenching of fluorescence by themetallic or semiconductor nanoparticles) (see FIG. 22). A negativeresult (no target nucleic acid present) is evidenced by fluorescencebecause the nanoparticles would pass through the pores of themicroporous material when it is washed (so no quenching of thefluorescence would occur) (see FIG. 22). There is low backgroundfluorescence because unbound probes are washed away from the detectionarea. In addition, in the case of a positive result, changes influorescence can be observed as a function of temperature. For instance,as the temperature is raised, fluorescence will be observed once thedehybridization temperature has been reached. Therefore, by looking atfluorescence as a function of temperature, information can be obtainedabout the degree of complementarity between the oligonucleotide probesand the target nucleic acid. Fluorescence can be generated by simplyilluminating the solution or microporous material with a UV lamp, andthe fluorescent signal can be monitored by the naked eye. Alternatively,for a more quantitative result, a fluorimeter can be employed infront-face mode to measure the fluorescence of the solution with a shortpath length.

[0194] In yet other embodiments, a “satellite probe” is used (see FIG.24). The satellite probe comprises a central particle with one orseveral physical properties that can be exploited for detection in anassay for nucleic acids (e.g., intense color, fluorescence quenchingability, magnetism). Suitable particles include the nanoparticles andother particles described above. The particle has oligonucleotides (allhaving the same sequence) attached to it (see FIG. 24). Methods ofattaching oligonucleotides to the particles are described above. Theseoligonucleotides comprise at least a first portion and a second portion,both of which are complementary to portions of the sequence of a targetnucleic acid (see FIG. 24). The satellite probe also comprises probeoligonucleotides. Each probe oligonucleotide has at least a firstportion and a second portion (see FIG. 24). The sequence of the firstportion of the probe oligonucleotides is complementary to the firstportion of the sequence of the oligonucleotides immobilized on thecentral particle (see FIG. 24). Consequently, when the central particleand the probe oligonucleotides are brought into contact, theoligonucleotides on the particle hybridize with the probeoligonucleotides to form the satellite probe (see FIG. 24). Both thefirst and second portions of the probe oligonucleotides arecomplementary to portions of the sequence of the target nucleic acid(see FIG. 24). Each probe oligonucleotide is labeled with a reportermolecule (see FIG. 24), as further described below. The amount ofhybridization overlap between the probe oligonucleotides and the target(length of the portion hybridized) is as large as, or greater than, thehybridization overlap between the probe oligonucleotides and theoligonucleotides attached to the particle (see FIG. 24). Therefore,temperature cycling resulting in dehybridization and rehybridizationwould favor moving the probe oligonucleotides from the central particleto the target. Then, the particles are separated from the probeoligonucleotides hybridized to the target, and the reporter molecule isdetected.

[0195] The satellite probe can be used in a variety of detectionstrategies. For example, if the central particle has a magnetic core andis covered with a materiai capable of quenching the fluorescence offluorophores attached to the probe oligonucleotides that surround it,this system can be used in an in situ fluorometric detection scheme fornucleic acids. Functionalized polymer-coated magnetic particles (Fe₃O₄)are available from several commercial sources including Dynal(Dynabeads™) and Bangs Laboratories (Estapor™), and silica-coatedmagnetic Fe₃O₄ nanoparticles could be modified (Liu et al., Chem.Mater., 10, 3936-3940 (1998))using well-developed silica surfacechemistry (Chrisey et al., Nucleic Acids Research, 24, 3031-3039 (1996))and employed as magnetic probes as well. Further, the dye molecule,4-((4-(dimethylamino)phenyl)-azo)benzoic acid (DABCYL) has been shown tobe an efficient quencher of fluorescence for a wide variety offluorphores attached to oligonucleotides (Tyagi et al., Nature Biotech.,16, 49-53 (1998). The commercially-available succinimidyl ester ofDABCYL (Molecular Probes) forms extremely stable amide bonds uponreaction with primary alkylamino groups. Thus, any magnetic particle orpolymer-coated magnetic particle with primary alkyl amino groups couldbe modified with both oligonucleotides, as well as these quenchermolecules. Alternatively, the DABCYL quencher could be attached directlyto the surface-bound oligonucleotide, instead of the alkylamino-modified surface. The satellite probe comprising the probeoligonucleotides is brought into contact with the target. Thetemperature is cycled so as to cause dehybridization andrehybridization, which causes the probe oligonucleotides to move fromthe central particle to the target. Detection is accomplished byapplying a magnetic field and removing the particles from solution andmeasuring the fluorescence of the probe oligonucleotides remaining insolution hybridized to the target.

[0196] This approach can be extended to a colorimetric assay by usingmagnetic particles with a dye coating in conjunction with probeoligonucleotides labeled with a dye which has optical properties thatare distinct from the dye on the magnetic nanoparticles or perturb thoseof the dye on the magnetic nanoparticles. When the particles and theprobe oligonucleotides are in solution together, the solution willexhibit one color which derives from a combination of the two dyes.However, in the presence of a target nucleic acid and with temperaturecycling, the probe oligonucleotides will move from the satellite probeto the target. Once this has happened, application of a magnetic fieldwill remove the magnetic, dye-coated particles from solution leavingbehind probe oligonucleotides labeled with a single dye hybridized tothe target. The system can be followed with a colorimeter or the nakedeye, depending upon target levels and color intensities.

[0197] This approach also can be further extended to an electrochemicalassay by using an oligonucleotide-magnetic particle conjugate inconjunction with a probe oligonucleotide having attached a redox-activemolecule. Any modifiable redox-active species can be used, such as thewell-studied redox-active ferrocene derivative. A ferrocene derivatizedphosphoramidite can be attached to oligonucleotides directly usingstandard phosphoramidite chemistry. Mucic et al., Chem. Commun., 555(1996); Eckstein, ed., in Oligonucleotides and Analogues, 1st ed.,Oxford University, New York, N.Y. (1991). The ferrocenylphosphoramiditeis prepared in a two-step synthesis from 6-bromohexylferrocene. In atypical preparation, 6-bromohexylferrocene is stirred in an aqueous HMPAsolution at 120° C. for 6 hours to from 6-hydroxyhexylferrocene. Afterpurification, the 6-hydroxyhexylferrocene is added to a THF solution ofN,N-diisopropylethylamine andbeta-cyanoethyl-N,N-diisopropylchlorophosphoramide to form theferrocenylphosphoramidite. Oligonucleotide-modified polymer-coated goldnanoparticles, where the polymer contains electrochemically-activeferrocene molecules, could also be utilized. Watson et al., J. Am. Chem.Soc., 121, 462-463 (1999). A copolymer of amino reactive sites (e.g.,anhydrides) could be incorporated into the polymer for reaction withamino-modified oligonucleotides. Moller et al., Bioconjugate Chem., 6,174-178 (1995). In the presence of target and with temperature cycling,the redox-active probe oligonucleotides will move from the satelliteprobe to the target. Once this has happened, application of the magneticfield will remove the magnetic particles from solution leaving behindthe redox-active probe oligonucleotides hybridized with the targetnucleic acid. The amount of target then can be determined by cyclicvoltammetry or any electrochemical technique that can interrogate theredox-active molecule.

[0198] In yet another embodiment of the invention, a nucleic acid isdetected by contacting the nucleic acid with a substrate havingoligonucleotides attached thereto. The oligonucleotides have a sequencecomplementary to a first portion of the sequence of the nucleic acid.The oligonucleotides are located between a pair of electrodes located onthe substrate. The substrate must be made of a material which is not aconductor of electricity (e.g., glass, quartz, polymers, plastics). Theelectrodes may be made of any standard material (e.g., metals, such asgold, platinum, tin oxide). The electrodes can be fabricated byconventional microfabrication techniques. See, e.g., Introduction ToMicrolithography (L. F. Thompson et al., eds., American ChemicalSociety, Washington, D.C. 1983). The substrate may have a plurality ofpairs of electrodes located on it in an array to allow for the detectionof multiple portions of a single nucleic acid, the detection of multipledifferent nucleic acids, or both. Arrays of electrodes can be purchased(e.g., from AbbtechScientific, Inc., Richmond, Va.) or can be made byconventional microfabrication techniques. See, e.g., Introduction ToMicrolithography (L. F. Thompson et al., eds., American ChemicalSociety, Washington, D.C. 1983). Suitable photomasks for making thearrays can be purchased (e.g., from Photronics, Milpitas, Calif.). Eachof the pairs of electrodes in the array will have a type ofoligonucleotides attached to the substrate between the two electrodes.The contacting takes place under conditions effective to allowhybridization of the oligonucleotides on the substrate with the nucleicacid. Then, the nucleic acid bound to the substrate, is contacted with atype of nanoparticles. The nanoparticles must be made of a materialwhich can conduct electricity. Such nanoparticles include those made ofmetal, such as gold nanoparticles, and semiconductor materials. Thenanoparticles will have one or more types of oligonucleotides attachedto them, at least one of the types of oligonucleotides having a sequencecomplementary to a second portion of the sequence of the nucleic acid.The contacting takes place under conditions effective to allowhybridization of the oligonucleotides on the nanoparticles with thenucleic acid. If the nucleic acid is present, the circuit between theelectrodes should be closed because of the attachment of thenanoparticles to the substrate between the electrodes, and a change inconductivity will be detected. If the binding of a single type ofnanoparticles does not result in closure of the circuit, this situationcan be remedied by using a closer spacing between the electrodes, usinglarger nanoparticles, or employing another material that will close thecircuit (but only if the nanoparticles have been bound to the substratebetween the electrodes). For instance, when gold nanoparticles are used,the substrate can be contacted with silver stain (as described above) todeposit silver between the electrodes to close the circuit and producethe detectable change in conductivity. Another way to close the circuitin the case where the addition of a single type of nanoparticles is notsufficient, is to contact the first type of nanoparticles bound to thesubstrate with a second type of nanoparticles having oligonucleotidesattached to them that have a sequence complementary to theoligonucleotides on the first type of nanoparticles. The contacting willtake place under conditions effective so that the oligonucleotides onthe second type of nanoparticle hybridize to those on the first type ofoligonucleotides. If needed, or desired, additional layers ofnanoparticles can be built up by alternately adding the first and secondtypes of nanoparticles until a sufficient number of nanoparticles areattached to the substrate to close the circuit. Another alternative tobuilding up individual layers of nanoparticles would be the use of anaggregate probe (see above).

[0199] The invention also provides kits for detecting nucleic acids. Inone embodiment, the kit comprises at least one container, the containerholding at least two types of nanoparticles having oligonucleotidesattached thereto. The oligonucleotides on the first type ofnanoparticles have a sequence complementary to the sequence of a firstportion of a nucleic acid. The oligonucleotides on the second type ofnanoparticles have a sequence complementary to the sequence of a secondportion of the nucleic acid. The container may further comprise filleroligonucleotides having a sequence complementary to a third portion ofthe nucleic acid, the third portion being located between the first andsecond portions. The filler oligonucleotide may also be provided in aseparate container.

[0200] In a second embodiment, the kit comprises at least twocontainers. The first container holds nanoparticles havingoligonucleotides attached thereto which have a sequence complementary tothe sequence of a first portion of a nucleic acid. The second containerholds nanoparticles having oligonucleotides attached thereto which havea sequence complementary to the sequence of a second portion of thenucleic acid. The kit may further comprise a third container holding afiller oligonucleotide having a sequence complementary to a thirdportion of the nucleic acid, the third portion being located between thefirst and second portions.

[0201] In another alternative embodiment, the kits can have theoligonucleotides and nanoparticles in separate containers, and theoligonucleotides would have to be attached to the nanoparticles prior toperforming an assay to detect a nucleic acid. The oligonucleotidesand/or the nanoparticles may be functionalized so that theoligonucleotides can be attached to the nanoparticles. Alternatively,the oligonucleotides and/or nanoparticles may be provided in the kitwithout functional groups, in which case they must be functionalizedprior to performing the assay.

[0202] In another embodiment, the kit comprises at least one container.The container holds metallic or semiconductor nanoparticles havingoligonucleotides attached thereto. The oligonucleotides have a sequencecomplementary to a portion of a nucleic acid and have fluorescentmolecules attached to the ends of the oligonucleotides not attached tothe nanoparticles.

[0203] In yet another embodiment, the kit comprises a substrate, thesubstrate having attached thereto nanoparticles. The nanoparticles haveoligonucleotides attached thereto which have a sequence complementary tothe sequence of a first portion of a nucleic acid. The kit also includesa first container holding nanoparticles having oligonucleotides attachedthereto which have a sequence complementary to the sequence of a secondportion of the nucleic acid. The oligonucleotides may have the same ordifferent sequences, but each of the oligonucleotides has a sequencecomplementary to a portion of the nucleic acid. The kit further includesa second container holding a binding oligonucleotide having a selectedsequence having at least two portions, the first portion beingcomplementary to at least a portion of the sequence of theoligonucleotides on the nanoparticles in the first container. The kitalso includes a third container holding nanoparticles havingoligonucleotides attached thereto, the oligonucleotides having asequence complementary to the sequence of a second portion of thebinding oligonucleotide.

[0204] In another embodiment, the kit comprises a substrate havingoligonucleotides attached thereto which have a sequence complementary tothe sequence of a first portion of a nucleic acid. The kit also includesa first container holding nanoparticles having oligonucleotides attachedthereto which have a sequence complementary to the sequence of a secondportion of the nucleic acid. The oligonucleotides may have the same ordifferent sequences, but each of the oligonucleotides has a sequencecomplementary to a portion of the nucleic acid. The kit further includesa second container holding nanoparticles having oligonucleotidesattached thereto which have a sequence complementary to at least aportion of the oligonucleotides attached to the nanoparticles in thefirst container.

[0205] In yet another embodiment, the kits can have the substrate,oligonucleotides and nanoparticles in separate containers. Thesubstrate, oligonucleotides, and nanoparticles would have to beappropriately attached to each other prior to performing an assay todetect a nucleic acid. The substrate, oligonucleotides and/or thenanoparticles may be functionalized to expedite this attachment.Alternatively, the substrate, oligonucleotides and/or nanoparticles maybe provided in the kit without functional groups, in which case theymust be functionalized prior to performing the assay.

[0206] In a further embodiment, the kit comprises a substrate havingoligonucleotides attached thereto which have a sequence complementary tothe sequence of a first portion of a nucleic acid. The kit also includesa first container holding liposomes having oligonucleotides attachedthereto which have a sequence complementary to the sequence of a secondportion of the nucleic acid and a second container holding nanoparticleshaving at least a first type of oligonucleotides attached thereto, thefirst type of oligonucleotides having a cholesteryl group attached tothe end not attached to the nanoparticles so that the nanoparticles canattach to the liposomes by hydrophobic interactions. The kit may furthercomprise a third container holding a second type of nanoparticles havingoligonucleotides attached thereto, the oligonucleotides having asequence complementary to at least a portion of the sequence of a secondtype of oligonucleotides attached to the first type of nanoparticles.The second type of oligonucleotides attached to the first type ofnanoparticles having a sequence complementary to the sequence of theoligonucleotides on the second type of nanoparticles.

[0207] In another embodiment, the kit may comprise a substrate havingnanoparticles attached to it. The nanoparticles have oligonucleotidesattached to them which have a sequence complementary to the sequence ofa first portion of a nucleic acid. The kit also includes a firstcontainer holding an aggregate probe. The aggregated probe comprises atleast two types of nanoparticles having oligonucleotides attached tothem. The nanoparticles of the aggregate probe are bound to each otheras a result of the hybridization of some of the oligonucleotidesattached to each of them. At least one of the types of nanoparticles ofthe aggregate probe has oligonucleotides attached to it which have asequence complementary to a second portion of the sequence of thenucleic acid.

[0208] In yet another embodiment, the kit may comprise a substratehaving oligonucleotides attached to it. The oligonucleotides have asequence complementary to the sequence of a first portion of a nucleicacid. The kit further includes a first container holding an aggregateprobe. The aggregate probe comprises at least two types of nanoparticleshaving oligonucleotides attached to them. The nanoparticles of theaggregate probe are bound to each other as a result of the hybridizationof some of the oligonucleotides attached to each of them. At least oneof the types of nanoparticles of the aggregate probe hasoligonucleotides attached thereto which have a sequence complementary toa second portion of the sequence of the nucleic acid.

[0209] In an additional embodiment, the kit may comprise a substratehaving oligonucleotides attached to it and a first container holding anaggregate probe. The aggregate probe comprises at least two types ofnanoparticles having oligonucleotides attached to them. Thenanoparticles of the aggregate probe are bound to each other as a resultof the hybridization of some of the oligonucleotides attached to each ofthem. At least one of the types of nanoparticles of the aggregate probehas oligonucleotides attached to it which have a sequence complementaryto a first portion of the sequence of the nucleic acid. The kit alsoincludes a second container holding nanoparticles. The nanoparticleshave at least two types of oligonucleotides attached to them. The firsttype of oligonucleotides has a sequence complementary to a secondportion of the sequence of the nucleic acid. The second type ofoligonucleotides has a sequence complementary to at least a portion ofthe sequence of the oligonucleotides attached to the substrate.

[0210] In another embodiment, the kit may comprise a substrate which hasoligonucleotides attached to it. The oligonucleotides have a sequencecomplementary to the sequence of a first portion of a nucleic acid. Thekit also comprises a first container holding liposomes havingoligonucleotides attached to them. The oligonucleotides have a sequencecomplementary to the sequence of a second portion of the nucleic acid.The kit further includes a second container holding an aggregate probecomprising at least two types of nanoparticles having oligonucleotidesattached to them. The nanoparticles of the aggregate probe are bound toeach other as a result of the hybridization of some of theoligonucleotides attached to each of them. At least one of the types ofnanoparticles of the aggregate probe has oligonucleotides attached to itwhich have a hydrophobic groups attached to the ends not attached to thenanoparticles.

[0211] In a further embodiment, the kit may comprise a first containerholding nanoparticles having oligonucleotides attached thereto. The kitalso includes one or more additional containers, each container holdinga binding oligonucleotide. Each binding oligonucleotide has a firstportion which has a sequence complementary to at least a portion of thesequence of oligonucleotides on the nanoparticles and a second portionwhich has a sequence complementary to the sequence of a portion of anucleic acid to be detected. The sequences is of the second portions ofthe binding oligonucleotides may be different as long as each sequenceis complementary to a portion of the sequence of the nucleic acid to bedetected.

[0212] In another embodiment, the kit comprises a container holding onetype of nanoparticles having oligonucleotides attached thereto and oneor more types of binding oligonucleotides. Each of the types of bindingoligonucleotides has a sequence comprising at least two portions. Thefirst portion is complementary to the sequence of the oligonucleotideson the nanoparticles, whereby the binding oligonucleotides arehybridized to the oligonucleotides on the nanoparticles in thecontainer(s). The second portion is complementary to the sequence of aportion of the nucleic acid.

[0213] In another embodiment, kits may comprise one or two containersholding two types of particles. The first type of particles havingoligonucleotides attached thereto which have a sequence complementary tothe sequence of a first portion of a nucleic acid. The oligonucleotidesare labeled with an energy donor on the ends not attached to theparticles. The second type of particles having oligonucleotides attachedthereto which have a sequence complementary to the sequence of a secondportion of a nucleic acid. The oligonucleotides are labeled with anenergy acceptor on the ends not attached to the particles. The energydonors and acceptors may be fluorescent molecules.

[0214] In a further embodiment, the kit comprises a first containerholding a type of latex microspheres having oligonucleotides attachedthereto. The oligonucleotides have a sequence complementary to a firstportion of the sequence of a nucleic acid and are labeled with afluorescent molecule. The kit also comprises a second container holdinga type of gold nanoparticles having oligonucleotides attached thereto.These oligonucleotides have a sequence complementary to a second portionof the sequence of the nucleic acid.

[0215] In another embodiment, the kit comprises a first containerholding a first type of metallic or semiconductor nanoparticles havingoligonucleotides attached thereto. The oligonucleotides have a sequencecomplementary to a first portion of the sequence of a nucleic acid andare labeled with a fluorescent molecule. The kit also comprises a secondcontainer holding a second type of metallic or semiconductornanoparticles having oligonucleotides attached thereto. Theseoligonucleotides have a sequence complementary to a second portion ofthe sequence of a nucleic acid and are labeled with a fluorescentmolecule.

[0216] In a further embodiment, the kit comprises a container holding asatellite probe. The satellite probe comprises a particle havingattached thereto oligonucleotides. The oligonucleotides have a firstportion and a second portion, both portions having sequencescomplementary to portions of the sequence of a nucleic acid. Thesatellite probe also comprises probe oligonucleotides hybridized to theoligonucleotides attached to the nanoparticles. The probeoligonucleotides have a first portion and a second portion. The firstportion has a sequence complementary to the sequence of the firstportion of the oligonucleotides attached to the particles, and bothportions have sequences complementary to portions of the sequence of thenucleic acid. The probe oligonucleotides also have a reporter moleculeattached to one end.

[0217] In another embodiment, the kit may comprise a container holdingan aggregate probe. The aggregate probe comprises at least two types ofnanoparticles having oligonucleotides attached to them. Thenanoparticles of the aggregate probe are bound to each other as a resultof the hybridization of some of the oligonucleotides attached to each ofthem. At least one of the types of nanoparticles of the aggregate probehas oligonucleotides attached to it which have a sequence complementaryto a portion of the sequence of a nucleic acid.

[0218] In an additional embodiment, the kit may comprise a containerholding an aggregate probe. The aggregate probe comprises at least twotypes of nanoparticles having oligonucleotides attached to them. Thenanoparticles of the aggregate probe are bound to each other as a resultof the hybridization of some of the oligonucleotides attached to each ofthem. At least one of the types of nanoparticles of the aggregate probehas oligonucleotides attached to it which have a hydrophobic groupattached to the end not attached to the nanoparticles.

[0219] In yet another embodiment, the invention provides a kitcomprising a substrate having located thereon at least one pair ofelectrodes with oligonucleotides attached to the substrate between theelectrodes. In a preferred embodiment, the substrate has a plurality ofpairs of electrodes attached to it in an array to allow for thedetection of multiple portions of a single nucleic acid, the detectionof multiple different nucleic acids, or both.

[0220] The kits may also contain other reagents and items useful fordetecting nucleic acid. The reagents may include PCR reagents, reagentsfor silver staining, hybridization reagents, buffers, etc. Other itemswhich may be provided as part of the kit include a solid surface (forvisualizing hybridization) such as a TLC silica plate, microporousmaterials, syringes, pipettes, cuvettes, containers, and a thermocycler(for controlling hybridization and de-hybridization temperatures).Reagents for functionalizing the nucleotides or nanoparticles may alsobe included in the kit.

[0221] The precipitation of aggregated nanoparticles provides a means ofseparating a selected nucleic acid from other nucleic acids. Thisseparation may be used as a step in the purification of the nucleicacid. Hybridization conditions are those described above for detecting anucleic acid. If the temperature is below the Tm (the temperature atwhich one-half of an oligonucleotide is bound to its complementarystrand) for the binding of the oligonucleotides on the nanoparticles tothe nucleic acid, then sufficient time is needed for the aggregate tosettle. The temperature of hybridization (e.g., as measured by Tm)varies with the type of salt (NaCl or MgCl₂) and its concentration. Saltcompositions and concentrations are selected to promote hybridization ofthe oligonucleotides on the nanoparticles to the nucleic acid atconvenient working temperatures without inducing aggregation of thecolloids in the absence of the nucleic acid.

[0222] The invention also provides a method of nanofabrication. Themethod comprises providing at least one type of linking oligonucleotidehaving a selected sequence. A linking oligonucleotide used fornanofabrication may have any desired sequence and may be single-strandedor double-stranded. It may also contain chemical modifications in thebase, sugar, or backbone sections. The sequences chosen for the linkingoligonucleotides and their lengths and strandedness will contribute tothe rigidity or flexibility of the resulting nanomaterial ornanostructure, or a portion of the nanomaterial or nanostructure. Theuse of a single type of linking oligonucleotide, as well as mixtures oftwo or more different types of linking oligonucleotides, iscontemplated. The number of different linking oligonucleotides used andtheir lengths will contribute to the shapes, pore sizes and otherstructural features of the resulting nanomaterials and nanostructures.

[0223] The sequence of a linking oligonucleotide will have at least afirst portion and a second portion for binding to oligonucleotides onnanoparticles. The first, second or more binding portions of the linkingoligonucleotide may have the same or different sequences.

[0224] If all of the binding portions of a linking oligonucleotide havethe same sequence, only a single type of nanoparticle witholigonucleotides having a complementary sequence attached thereto needbe used to form a nanomaterial or nanostructure. If the two or morebinding portions of a linking oligonucleotide have different sequences,then two or more nanoparticle-oligonucleotide conjugates must be used.See, e.g., FIG. 17. The oligonucleotides on each of the nanoparticleswill have a sequence complementary to one of the two or more bindingportions of the sequence of the linking oligonucleotide The number,sequence(s) and length(s) of the binding portions and the distance(s),if any, between them will contribute to the structural and physicalproperties of the resulting nanomaterials and nanostructures. Of course,if the linking oligonucleotide comprises two or more portions, thesequences of the binding portions must be chosen so that they are notcomplementary to each other to avoid having one portion of the linkingnucleotide bind to another portion.

[0225] The linking oligonucleotides and nanoparticle-oligonucleotideconjugates are contacted under conditions effective for hybridization ofthe oligonucleotides attached to the nanoparticles with the linkingoligonucleotides so that a desired nanomaterial or nanostructure isformed wherein the nanoparticles are held together by oligonucleotideconnectors. These hybridization conditions are well known in the art andcan be optimized for a particular nanofabrication scheme (see above).Stringent hybridization conditions are preferred.

[0226] The invention also provides another method of nanofabrication.This method is comprises providing at least two types ofnanoparticle-oligonucleotide conjugates. The oligonucleotides on thefirst type of nanoparticles have a sequence complementary to that of theoligonucleotides on the second type of nanoparticles. Theoligonucleotides on the second type of nanoparticles have a sequencecomplementary to that of the oligonucleotides on the first type ofnanoparticles. The nanoparticle-oligonucleotide conjugates are contactedunder conditions effective to allow hybridization of theoligonucleotides on the nanoparticles to each other so that a desirednanomaterial or nanostructure is formed wherein the nanoparticles areheld together by oligonucleotide connectors. Again, these hybridizationconditions are well-known in the art and can be optimized for aparticular nanofabrication scheme.

[0227] In both nanofabrication methods of the invention, the use ofnanoparticles having one or more different types of oligonucleotidesattached thereto is contemplated. The number of differentoligonucleotides attached to a nanoparticle and the lengths andsequences of the one or more oligonucleotides will contribute to therigidity and structural features of the resulting nanomaterials andnanostructures.

[0228] Also, the size, shape and chemical composition of thenanoparticles will contribute to the properties of the resultingnanomaterials and nanostructures. These properties include opticalproperties, optoelectronic properties, electrochemical properties,electronic properties, stability in various solutions, pore and channelsize variation, ability to separate bioactive molecules while acting asa filter, etc. The use of mixtures of nanoparticles having differentsizes, shapes and/or chemical compositions, as well as the use ofnanoparticles having uniform sizes, shapes and chemical composition, arecontemplated.

[0229] In either fabrication method, the nanoparticles in the resultingnanomaterial or nanostructure are held together by oligonucleotideconnectors. The sequences, lengths, and strandedness of theoligonucleotide connectors, and the number of different oligonucleotideconnectors present will contribute to the rigidity and structuralproperties of the nanomaterial or nanostructure. If an oligonucleotideconnector is partially double-stranded, its rigidity can be increased bythe use of a filler oligonucleotide as described above in connectionwith the method of detecting nucleic acid. The rigidity of a completelydouble-stranded oligonucleotide connector can be increased by the use ofone or more reinforcing oligonucleotides having complementary sequencesso that they bind to the double-stranded oligonucleotide connector toform triple-stranded oligonucleotide connectors. The use ofquadruple-stranded oligonucleotide connectors based on deoxyquanosine ordeoxycytidine quartets is also contemplated.

[0230] Several ofa variety of systems for organizing nanoparticles basedon oligonucleotide hybridization are illustrated in the figures. In asimple system (FIG. 1) one set of nanoparticles bears oligonucleotideswith a defined sequence and another set ofnanoparticles bearsoligonucleotides with a complementary sequence. On mixing the two setsof nanoparticle-oligonucleotide conjugates under hybridizationconditions, the two types of particles are linked by double strandedoligonucleotide connectors which serve as spacers to position thenanoparticles at selected distances.

[0231] An attractive system for spacing nanoparticles involves theaddition of one free linking oligonucleotide as illustrated in FIG. 2.The sequence of the linking oligonucleotide will have at least a firstportion and a second portion for binding to oligonucleotides onnanoparticles. This system is basically the same as utilized in thenucleic acid detection method, except that the length of the addedlinking oligonucleotide can be selected to be equal to the combinedlengths of oligonucleotides attached to the nanoparticles. The relatedsystem illustrated in FIG. 3 provides a convenient means to tailor thedistance between nanoparticles without having to change the sets ofnanoparticle-oligonucleotide conjugates employed.

[0232] A further elaboration of the scheme for creating defined spacesbetween nanoparticles is illustrated in FIG. 4. In this case a doublestranded segment of DNA or RNA containing overhanging ends is employedas the linking oligonucleotide. Hybridization of the single-stranded,overhanging segments of the linking oligonucleotide with theoligonucleotides attached to the nanoparticles affords multipledouble-stranded oligonucleotide cross-links is between thenanoparticles.

[0233] Stiffer nanomaterials and nanostructures, or portions thereof,can be generated by employing triple-stranded oligonucleotide connectorsbetween nanoparticles. In forming the triple strand, one may exploiteither the pyrimidine:purine:pyrimidine motif (Moser, H. E. and Dervan,P. B. Science, 238,645-650 (1987) or the purine:purine:pyrimidinemotif(Pilch, D. S. et al. Biochemistry, 30, 6081-6087(1991). An exampleof the organization ofnanoparticles by generating triple-strandedconnectors by the pyrimidine:purine:pyrimidine motif are illustrated inFIG. 10. In the system shown in FIG. 10, one set of nanoparticles isconjugated with a defined strand containing pyrimidine nucleotides andthe other set is conjugated with a complementary oligonucleotidecontaining purine nucleotides. Attachment of the oligonucleotides isdesigned such that the nanoparticles are separated by thedouble-stranded oligonucleotide formed on hybridization. Then, a freepyrimidine oligonucleotide with an orientation opposite that for thepyrimidine strand linked to the nanoparticle is added to the systemprior to, simultaneously with, or just subsequent to mixing thenanoparticles. Since the third strand in this system is held byHoogsteen base pairing, the triple strand is relatively unstablethermally. Covalent bridges spanning the breadth of the duplex are knownto stabilize triple-stranded complexes (Salunke, M., Wu, T., Letsinger,R. L., J. Am, Chem. Soc. 114, 8768-8772, (1992). Letsinger, R. L. andWu, T. J. Am Chem. Soc., 117, 7323-7328 (1995). Prakash, G. and Kool, J.Am. Chem. Soc., 114, 3523-3527 (1992).

[0234] For construction of nanomaterials and nanostructures, it may bedesirable in some cases to “lock” the assembly in place by covalentcross-links after formation of the nanomaterial or nanostructure byhybridization of the oligonucleotide components. This can beaccomplished by incorporating functional groups that undergo a triggeredirreversible reaction into the oligonucleotides. An example of afunctional group for this purpose is a stilbenedicarboxamide group. Ithas been demonstrated that two stilbenedicarboxamide groups alignedwithin hybridized oligonucleotides readily undergo cross-linking onirradiation with ultraviolet light (340 nm) (Lewis, F. D. et al. (1995)J. Am. Chem. Soc. 117, 8785-8792).

[0235] Alternatively, one could employ the displacement of a 5′-O-tosylgroup from an oligonucleotide, held at the 3′-position to a nanoparticleby a mercaptoalkly group, with a thiophosphoryl group at the 3′-end ofan oligonucleotide held to an nanoparticle by a mercaptoalkyl group. Inthe presence of an oligonucleotide that hybridizes to botholigonucleotides and, thereby, brings the thiophosphoryl group intoproximity of the tosyl group, the tosyl group will be displaced by thethiophosphoryl group, generating an oligonucleotide linked at the endsto two different nanoparticles. For displacement reactions ofthis type,see Herrlein et al., J. Am. Chem. Soc., 177, 10151-10152 (1995). Thefact that thiophosphoryl oligonucleotides do not react with goldnanoparticles under the conditions employed in attachingmercaptoalkyl-oligonucleotides to gold nanoparticles enables one toprepare gold nanoparticle-oligonucleotide conjugates anchored throughthe mercapto group to the nanoparticles and containing a terminalthiophosphoryl group free for the coupling reaction.

[0236] A related coupling reaction to lock the assembled nanoparticlesystem in place utilizes displacement of bromide from a terminalbromoacetylaminonucleoside by a terminal thiophosphoryl-oligonucleotideas described in Gryaznov and Letsinger, J. Am. Chem. Soc., 115, 3808.This reaction proceeds much like the displacement of tosylate describedabove, except that the reaction is faster. Nanoparticles bearingoligonucleotides terminated with thiophosphoryl groups are prepared asdescribed above. For preparation of nanoparticles bearingoligonucleotides terminated with bromoacetylamino groups, one firstprepares an oligonucleotide terminated at one end by an aminonucleoside(e.g., either 5′-amino-5′-deoxythymidine or 3′-amino-3′-deoxythymidine)and at the other end by a mercaptoalkyl group. Molecules of thisoligonucleotide are then anchored to the nanoparticles through themercapto groups, and the nanoparticle-oligonucleotide conjugate is thenconverted the N-bromoacetylamino derivative by reaction with abromoacetyl acylating agent.

[0237] A fourth coupling scheme to lock the assemblies in place utilizesoxidation of nanoparticles bearing oligonucleoti des terminated bythiophosphoryl groups. Mild oxidizing agents, such as potassiumtriiodide, potassium ferricyanide (see Gryaznov and Letsinger, NucleicAcids Research, 21, 1403) or oxygen, are preferred.

[0238] In addition, the properties of the nanomaterials andnanostructures can be altered by incorporating into the interconnectingoligonucleotide chains organic and inorganic functions that are held inplace by covalent attachment to the oligonucleotide chains. A widevariety of backbone, base and sugar modifications are well known (seefor example Uhlmann, E., and Peyman, A. Chemical Reviews, 90, 544-584(1990). Also, the oligonucleotide chains could be replaced by “PeptideNucleic Acid” chains (PNA), in which the nucleotide bases are held by apolypeptide backbone (see Wittung, P. et al., Nature, 368, 561-563(1994).

[0239] As can be seen from the foregoing, the nanofabrication method ofthe invention is extremely versatile. By varying the length, sequenceand strandedness of the linking oligonucleotides, the number, length,and sequence of the binding portions of the linking oligonucleotides,the length, sequence and number of the oligonucleotides attached to thenanoparticles, the size, shape and chemical composition of thenanoparticles, the number and types of different linkingoligonucleotides and nanoparticles used, and the strandedness of theoligonucleotide connectors, nanomaterials and nanostructures having awide range of structures and properties can be prepared. Thesestructures and properties can be varied further by cross-linking of theoligonucleotide connectors, by functionalizing the oligonucleotides, bybackbone, base or sugar modifications of the oligonucleotides, or by theuse of peptide-nucleic acids.

[0240] The nanomaterials and nanostructures that can be made by thenanofabrication method of the invention include nanoscale mechanicaldevices, separation membranes, bio-filters, and biochips. It iscontemplated that the nanomaterials and nanostructures of the inventioncan be used as chemical sensors, in computers, for drug delivery, forprotein engineering, and as templates for biosynthesis/nanostructurefabrication/directed assembly of other structures. See generally Seemanet al., New J. Chem., 17, 739 (1993) for other possible applications.The nanomaterials and nanostructures that can be made by thenanofabrication method of the invention also can include electronicdevices. Whether nucleic acids could transport electrons has been thesubject of substantial controversy. As shown in Example 21 below,nanoparticles assembled by DNA conduct electricity (the DNA connectorsfunction as semiconductors).

[0241] Finally, the invention provides methods of making uniquenanoparticle-oligonucleotide conjugates. In the first such method,oligonucleotides are bound to charged nanoparticles to produce stablenanoparticle-oligonucleotide conjugates. Charged nanoparticles includenanoparticles made of metal, such as gold nanoparticles.

[0242] The method comprises providing oligonucleotides having covalentlybound thereto a moiety comprising a functional group which can bind tothe nanoparticles. The moieties and functional groups are thosedescribed above for binding (i.e., by chemisorption or covalent bonding)oligonucleotides to nanoparticles. For instance, oligonucleotides havingan alkanethiol or an alkanedisulfide covalently bound to their 5′ or 3′ends can be used to bind the oligonucleotides to a variety ofnanoparticles, including gold nanoparticles.

[0243] The oligonucleotides are contacted with the nanoparticles inwater for a time sufficient to allow at least some of theoligonucleotides to bind to the nanoparticles by means of the functionalgroups. Such times can be determined empirically. For instance, it hasbeen found that a time of about 12-24 hours gives good results. Othersuitable conditions for binding of the oligonucleotides can also bedetermined empirically. For instance, a concentration of about 10-20 nMnanoparticles and incubation at room temperature gives good results.

[0244] Next, at least one salt is added to the water to form a saltsolution. The salt can be any water-soluble salt. For instance, the saltmay be sodium chloride, magnesium chloride, potassium chloride, ammoniumchloride, sodium acetate, ammonium acetate, a combination of two or moreof these salts, or one of these salts in phosphate buffer. Preferably,the salt is added as a concentrated solution, but it could be added as asolid. The salt can be added to the water all at one time or the salt isadded gradually over time. By “gradually over time” is meant that thesalt is added in at least two portions at intervals spaced apart by aperiod of time. Suitable time intervals can be determined empirically.

[0245] The ionic strength of the salt solution must be sufficient toovercome at least partially the electrostatic repulsion of theoligonucleotides from each other and, either the electrostaticattraction of the negatively-charged oligonucleotides forpositively-charged nanoparticles, or the electrostatic repulsion of thenegatively-charged oligonucleotides from negatively-chargednanoparticles. Gradually reducing the electrostatic attraction andrepulsion by adding the salt gradually over time has been found to givethe highest surface density of oligonucleotides on the nanoparticles.Suitable ionic strengths can be determined empirically for each salt orcombination of salts. A final concentration of sodium chloride of fromabout 0.1 M to about 1.0 M in phosphate buffer, preferably with theconcentration of sodium chloride being increased gradually over time,has been found to give good results.

[0246] After adding the salt, the oligonucleotides and nanoparticles areincubated in the salt solution for an additional period of timesufficient to allow sufficient additional oligonucleotides to bind tothe nanoparticles to produce the stable nanoparticle-oligonucleotideconjugates. As will be described in detail below, an increased surfacedensity of the oligonucleotides on the nanoparticles has been found tostabilize the conjugates. The time of this incubation can be determinedempirically. A total incubation time of about 24-48, preferably 40hours, has been found to give good results (this is the total time ofincubation; as noted above, the salt concentration can be increasedgradually over this total time). This second period of incubation in thesalt solution is referred to herein as the “aging” step. Other suitableconditions for this “aging” step can also be determined empirically. Forinstance, incubation at room temperature and pH 7.0 gives good results.

[0247] The conjugates produced by use of the “aging” step have beenfound to be considerably more stable than those produced without the“aging” step. As noted above, this increased stability is due to theincreased density of the oligonucleotides on the surfaces of thenanoparticles which is achieved by the “aging” step. The surface densityachieved by the “aging” step will depend on the size and type ofnanoparticles and on the length, sequence and concentration of theoligonucleotides. A surface density adequate to make the nanoparticlesstable and the conditions necessary to obtain it for a desiredcombination of nanoparticles and oligonucleotides can be determinedempirically. Generally, a surface density of at least 10 picomoles/cm²will be adequate to provide stable nanoparticle-oligonucleotideconjugates. Preferably, the surface density is at least 15picomoles/cm². Since the ability of the oligonucleotides of theconjugates to hybridize with nucleic acid and oligonucleotide targetscan be diminished if the surface density is too great, the surfacedensity is preferably no greater than about 35-40 picomoles/cm².

[0248] As used herein, “stable” means that, for a period of at least sixmonths after the conjugates are made, a majority of the oligonucleotidesremain attached to the nanoparticles and the oligonucleotides are ableto hybridize with nucleic acid and oligonucleotide targets understandard conditions encountered in methods of detecting nucleic acid andmethods of nanofabrication.

[0249] Aside from their stability, the nanoparticle-oligonucleotideconjugates made by this method exhibit other remarkable properties. See,e.g., Examples 5, 7, and 19 of the present application. In particular,due to the high surface density of the conjugates, they will assembleinto large aggregates in the presence of a target nucleic acid oroligonucleotide. The temperature over which the aggregates form anddissociate has unexpectedly been found to be quite narrow, and thisunique feature has important practical consequences. In particular, itincreases the selectivity and sensitivity of the methods of detection ofthe present invention. A single base mismatch and as little as 20femtomoles of target can be detected using the conjugates. Althoughthese features were originally discovered in assays performed insolution, the advantages of the use of these conjugates have been foundto extend to assays performed on substrates, including those in whichonly a single type of conjugate is used.

[0250] It has been found that the hybridization efficiency ofnanoparticle-oligonucleotide conjugates can be increased dramatically bythe use of recognition oligonucleotides which comprise a recognitionportion and a spacer portion. “Recognition oligonucleotides” areoligonucleotides which comprise a sequence complementary to at least aportion of the sequence of a nucleic acid or oligonucleotide target. Inthis embodiment, the recognition oligonucleotides comprise a recognitionportion and a spacer portion, and it is the recognition portion whichhybridizes to the nucleic acid or oligonucleotide target. The spacerportion of the recognition oligonucleotide is designed so that it canbind to the nanoparticles. For instance, the spacer portion could have amoiety covalently bound to it, the moiety comprising a functional groupwhich can bind to the nanoparticles. These are the same moieties andfunctional groups as described above. As a result of the binding of thespacer portion of the recognition oligonucleotide to the nanoparticles,the recognition portion is spaced away from the surface of thenanoparticles and is more accessible for hybridization with its target.The length and sequence of the spacer portion providing good spacing ofthe recognition portion away from the nanoparticles can be determinedempirically. It has been found that a spacer portion comprising at leastabout 10 nucleotides, preferably 10-30 nucleotides, gives good results.The spacer portion may have any sequence which does not interfere withthe ability of the recognition oligonucleotides to become bound to thenanoparticles or to a nucleic acid or oligonucleotide target. Forinstance, the spacer portions should not sequences complementary to eachother, to that of the recognition olignucleotides, or to that of thenucleic acid or oligonucleotide target of the recognitionoligonucleotides. Preferably, the bases of the nucleotides of the spacerportion are all adenines, all thymines, all cytidines, or all guanines,unless this would cause one of the problems just mentioned. Morepreferably, the bases are all adenines or all thymines. Most preferablythe bases are all thymines.

[0251] It has further been found that the use of diluentoligonucleotides in addition to recognition oligonucleotides provides ameans of tailoring the conjugates to give a desired level ofhybridization. The diluent and recognition oligonucleotides have beenfound to attach to the nanoparticles in about the same proportion astheir ratio in the solution contacted with the nanoparticles to preparethe conjugates. Thus, the ratio of the diluent to recognitionoligonucleotides bound to the nanoparticles can be controlled so thatthe conjugates will participate in a desired number of hybridizationevents. The diluent oligonucleotides may have any sequence which doesnot interfere with the ability of the recognition oligonucleotides to bebound to the nanoparticles or to bind to a nucleic acid oroligonucleotide target. For instance, the diluent oligonulceotidesshould not have a sequence complementary to that of the recognitionolignucleotides or to that of the nucleic acid or oligonucleotide targetof the recognition oligonucleotides. The diluent oligonucleotides arealso preferably of a length shorter than that of the recognitionoligonucleotides so that the recognition oligonucleotides can bind totheir nucleic acid or oligonucleotide targets. If the recognitionoligonucleotides comprise spacer portions, the diluent oligonulceotidesare, most preferably, about the same length as the spacer portions. Inthis manner, the diluent oligonucleotides do not interefere with theability of the recognition portions of the recognition oligonucleotidesto hybridize with nucleic acid or oligonucleotide targets. Even morepreferably, the diluent oligonucleotides have the same sequence as thesequence of the spacer portions of the recognition oligonucleotides.

[0252] As can be readily appreciated, highly desirablenanoparticle-oligonucleotide conjugates can be prepared by employing allof the methods described above. By doing so, stable conjugates withtailored hybridization abilities can be produced.

[0253] Any of the above conjugates can be, and are preferably, used inany of the methods of detecting nucleic acids described above, and theinvention also provides a kit comprising a container holding any of theabove conjugates. In addition, the conjugates can be, and arepreferably, used in any of the methods of nanofabrication of theinvention and the method of separating nucleic acids.

[0254] It is to be noted that the term “a” or “an” entity refers to oneor more of that entity. For example, “a characteristic” refers to one ormore characteristics or at least one characteristic. As such, the terms“a” (or “an”), “one or more” and “at least one” are used interchangeablyherein. It is also to be noted that the terms “comprising”, “including”,and “having” have been used interchangeably.

EXAMPLES Example 1 Preparation of Oligonucleotide-modified GoldNanoparticles A. Preparation Of Gold Nanoparticles

[0255] Gold colloids (13 nm diameter) were prepared by reduction ofHAuCl₄ with citrate as described in Frens, Nature Phys. Sci., 241, 20(1973) and Grabar, Anal. Chem., 67, 735 (1995). Briefly, all glasswarewas cleaned in aqua regia (3 parts HCl, 1 part HNO₃), rinsed withNanopure H₂O, then oven dried prior to use. HAuCl₄ and sodium citratewere purchased from Aldrich Chemical Company. Aqueous HAuCl₄ (1 mM, 500mL) was brought to reflux while stirring. Then, 38.8 mM sodium citrate(50 mL) was added quickly. The solution color changed from pale yellowto burgundy, and refluxing was continued for 15 min. After cooling toroom temperature, the red solution was filtered through a MicronSeparations Inc. 1 micron filter. Au colloids were characterized byUV-vis spectroscopy using a Hewlett Packard 8452A diode arrayspectrophotometer and by Transmission Electron Microscopy (TEM) using aHitachi 8100 transmission electron microscope. Gold particles withdiameters of 13 nm will produce a visible color change when aggregatedwith target and probe oligonucleotide sequences in the 10-35 nucleotiderange.

B. Synthesis Of Oligonucleotides

[0256] Oligonucleotides were synthesized on a 1 micromole scale using aMilligene Expedite DNA synthesizer in single column mode usingphosphoramidite chemistry. Eckstein, F. (ed.) Oligonucleotides andAnalogues: A Practical Approach (IRL Press, Oxford, 1991). All solutionswere purchased from Milligene (DNA synthesis grade). Average couplingefficiency varied from 98 to 99.8%, and the final dimethoxytrityl (DMT)protecting group was not cleaved from the oligonucleotides to aid inpurification.

[0257] For 3′-thiol-oligonucleotides, Thiol-Modifier C3 S-S CPG supportwas purchased from Glen Research and used in the automated synthesizer.During normal cleavage from the solid support (16 hr at 55° C.), 0.05 Mdithiothreitol (DTT) was added to the NH₄OH solution to reduce the 3′disulfide to the thiol. Before purification by reverse phase highpressure liquid chromatography (HPLC), excess DTT was removed byextraction with ethyl acetate.

[0258] For 5′-thiol oligonucleotides, 5′-Thiol-ModifierC₆-phosphoramidite reagent was purchased from Glen Research, 44901Falcon Place, Sterling, Va. 20166. The oligonucleotides weresynthesized, and the final DMT protecting group removed. Then, 1 ml ofdry acetonitrile was added to 100 μmole of the 5′ Thiol ModifierC₆-phosphoramidite. 200 μL of the amidite solution and 200 μL ofactivator (fresh from synthesizer) were mixed and introduced onto thecolumn containing the synthesized oligonucleotides still on the solidsupport by syringe and pumped back and forth through the column for 10minutes. The support was then washed (2×1 mL) with dry acetonitrile for30 seconds. 700 μL of a 0.016 M I₂/H₂O/pyridine mixture (oxidizersolution) was introduced into the column, and was then pumped back andforth through the column with two syringes for 30 second. The supportwas then washed with a 1:1 mixture of CH₃CN/pyridine (2×1 mL) for 1minute, followed by a final wash with dry acetonitrile (2×1 mL) withsubsequent drying of the column with a stream of nitrogen. The tritylprotecting group was not removed, which aids in purification.

[0259] Reverse phase HPLC was performed with a Dionex DX500 systemequipped with a Hewlett Packard ODS hypersil column (4.6×200 mm, 5 mmparticle size) using 0.03 M Et₃NH⁺ OAc⁻ buffer (TEAA), pH 7, with a1%/min. gradient of 95% CH₃CN/5% TEAA. The flow rate was 1 mL/min. withUV detection at 260 nm. Preparative HPLC was used to purify theDMT-protected unmodified oligonucleotides (elution at 27 min). Aftercollection and evaporation of the buffer, the DMT was cleaved from theoligonucleotides by treatment with 80% acetic acid for 30 min at roomtemperature. The solution was then evaporated to near dryness, water wasadded, and the cleaved DMT was extracted from the aqueousoligonucleotide solution using ethyl acetate. The amount ofoligonucleotide was determined by absorbance at 260 nm, and final purityassessed by reverse phase HPLC (elution time 14.5 minutes).

[0260] The same protocol was used for purification of the3′-thiol-oligonucleotides, except that DTT was added after extraction ofDMT to reduce the amount of disulfide formed. After six hours at 40° C.,the DTT was extracted using ethyl acetate, and the oligonucleotidesrepurified by HPLC (elution time 15 minutes).

[0261] For purification of the 5′ thiol modified oligonucleotides,preparatory HPLC was performed under the same conditions as forunmodified oligonucleotides. After purification, the trityl protectinggroup was removed by adding 150 μL of a 50 mM AgNO₃ solution to the dryoligonucleotide sample. The sample turned a milky white color as thecleavage occurred. After 20 minutes, 200 μL of a 10 mg/ml solution ofDTT was added to complex the Ag (five minute reaction time), and thesample was centrifuged to precipitate the yellow complex. Theoligonucleotide solution (<50 OD) was then transferred onto a desaltingNAP-5 column (Pharmacia Biotech, Uppsala, Sweden) for purification(contains DNA Grade Sephadex G-25 Medium for desalting and bufferexchange of oligonucleotides greater than 10 bases). The amount of 5′thiol modified oligonucleotide was determined by UV-vis spectroscopy bymeasuring the magnitude of the absorbance at 260 nm. The final puritywas assessed by performing ion-exchange HPLC with a Dionex NucleopacPA-100 (4×250) column using a 10 mM NaOH solution (pH 12) with a 2%/mingradient of 10 mM NaOH, 1 M NaCl solution. Typically, two peaks resultedwith elution times of approximately 19 minutes and 25 minutes (elutiontimes are dependent on the length of the oligonucleotide strand). Thesepeaks corresponded to the thiol and the disulfide oligonucleotidesrespectively.

C. Attachment of Oligonucleotides to Gold Nanoparticles

[0262] An aqueous solution of 17 nM (150 μL) Au colloids, prepared asdescribed in part A above, was mixed with 3.75 μM (46 μL)3′-thiol-TTTGCTGA, prepared as described in part B and allowed to standfor 24 hours at room temperature in 1 ml Eppendorf capped vials. Asecond solution of colloids was reacted with 3.75 μM (46 μL)3′-thiol-TACCGTTG. Note that these oligonucleotides arenoncomplementary. Shortly before use, equal amounts of each of the twonanoparticle solutions were combined. Since the oligonucleotides arenoncomplementary, no reaction took place.

[0263] The oligonucleotide-modified nanoparticles are stable at elevatedtemperatures (80° C.) and high salt concentrations (1M NaCl) for daysand have not been observed to undergo particle growth. Stability in highsalt concentrations is important, since such conditions are required forthe hybridization reactions that form the basis of the methods ofdetection and nanofabrication of the invention.

Example 2 Formation of Nanoparticle Aggregates A. Preparation Of LinkingOligonucleotide

[0264] Two (nonthiolated) oligonucleotides were synthesized as describedin part B of Example 1. They had the following sequences: 3′ ATATGCGCGATCTCAGCAAA [SEQ ID NO:1]; and 3′ GATCGCGCAT ATCAACGGTA [SEQ ID NO:2].

[0265] Mixing of these two oligonucleotides in a 1 M NaCl, 10 mMphosphate buffered (pH 7.0) solution, resulted in hybridization to forma duplex having a 12-base-pair overlap and is two 8-base-pair stickyends. Each of the sticky ends had a sequence which was complementary tothat of one of the oligonucleotides attached to the Au colloids preparedin part C of Example 1.

B. Formation Of Nanoparticle Aggregates

[0266] The linking oligonucleotides prepared in part A of this example(0.17 μM final concentration after dilution with NaCl) were added to thenanoparticle-oligonucleotide conjugates prepared in part C of Example 1(5.1 mM final concentration after dilution with NaCl) at roomtemperature. The solution was then diluted with aqueous NaCl (to a finalconcentration of 1 M) and buffered at pH 7 with 10 mM phosphate,conditions which are suitable for hybridization of the oligonucleotides.An immediate color change from red to purple was observed, and aprecipitation reaction ensued. See FIG. 6. Over the course of severalhours, the solution became clear and a pinkish-gray precipitate settledto the bottom of the reaction vessel. See FIG. 6.

[0267] To verify that this process involved both the oligonucleotidesand colloids, the precipitate was collected and resuspended (by shaking)in 1 M aqueous NaCl buffered at pH 7. Any of the oligonucleotides nothybridized to the nanoparticles are removed in this manner. Then, atemperature/time dissociation experiment was performed by monitoring thecharacteristic absorbance for the hybridized oligodeoxyribonucleotides(260 nm) and for the aggregated colloids which is reflective of the goldinterparticle distance (700 nm). See FIG. 7.

[0268] Changes in absorbance at 260 and 700 nm were recorded on aPerkin-Elmer Lambda 2 UV-vis Spectrophotometer using a Peltier PTP-1Temperature Controlled Cell Holder while cycling the temperature at arate of 1° C./minute between 0° C. and 80° C. DNA solutions wereapproximately 1 absorbance unit(s) (OD), buffered at pH 7 using 10 mMphosphate buffer and at 1M NaCl concentration.

[0269] The results are shown in FIG. 8A. As the temperature was cycledbetween 0° C. and 80° C. (which is 38° C. above the dissociationtemperature (T_(m)) for the duplex (T_(m)=42° C.)), there was anexcellent correlation between the optical signatures for both thecolloids and oligonucleotides. The UV-vis spectrum for naked Au colloidswas much less temperature dependent, FIG. 8B.

[0270] There was a substantial visible optical change when the polymericoligonucleotide-colloid precipitate was heated above its melting point.The clear solution turned dark red as the polymeric biomaterialde-hybridized to generate the unlinked colloids which are soluble in theaqueous solution. The process was reversible, as evidenced by thetemperature traces in FIG. 8A.

[0271] In a control experiment, a 14-T:14-A duplex was shown to beineffective at inducing reversible Au colloid particle aggregation. Inanother control experiment, a linking oligonucleotide duplex with fourbase pair mismatches in the sticky ends was found not to inducereversible particle aggregation of oligonucleotide-modifiednanoparticles (prepared as described in part C of Example 1 and reactedas described above). In a third control experiment, non-thiolatedoligonucleotides having sequences complementary to the sticky ends ofthe linking oligonucleotide and reacted with nanoparticles did notproduce reversible aggregation when the nanoparticles were combined withthe linking oligonucleotide.

[0272] Further evidence of the polymerization/assembly process came fromTransmission Electron Microscopy (TEM) studies of the precipitate. TEMwas performed on a Hitachi 8100 Transmission Electron Microscope. Atypical sample was prepared by dropping 100 μL of colloid solution ontoa holey carbon grid. The grid, then, was dried under vacuum and imaged.TEM images of Au colloids linked by hybridized oligonucleotides showedlarge assembled networks of the Au colloids, FIG. 9A. Naked Au colloidsdo not aggregate under comparable conditions but rather disperse orundergo particle growth reactions. Hayat, Colloidal Gold: Principles,Methods, and Applications (Academic Press, San Diego, 1991). Note thatthere is no evidence of colloid particle growth in the experimentsperformed to date; the hybridized colloids seem to be remarkably regularin size with an average diameter of 13 nm.

[0273] With TEM, a superposition of layers is obtained, making itdifficult to assess the degree of order for three-dimensionalaggregates. However, smaller scale images of single layer,two-dimensional aggregates provided more evidence for the self-assemblyprocess, FIG. 9B. Close-packed assemblies of the aggregates with uniformparticle separations of approximately 60 Å can be seen. This distance issomewhat shorter than the estimated 95 Å spacing expected for colloidsconnected by rigid oligonucleotide hybrids with the sequences that wereused. However, because of the nicks in the duplex obtained afterhybridization of the oligonucleotides on the nanoparticles to thelinking oligonucleotides, these were not rigid hybrids and were quiteflexible. It should be noted that this is a variable that can becontrolled by reducing the system from four overlapping strands to three(thereby reducing the number of nicks) or by using triplexes instead ofduplexes.

Example 3 Preparation of Oligonucleotide-modified Gold Nanoparticles

[0274] Gold colloids (13 nm diameter) were prepared as described inExample 1. Thiol-oligonucleotides [HS(CH₂)₆OP(O)(O⁻)-oligonucleotide]were also prepared as described in Example 1.

[0275] The method of attaching thiol-oligonucleotides to goldnanoparticles described in Example 1 was found not to producesatisfactory results in some cases. In particular, when longoligonucleotides were used, the oligonucleotide-colloid conjugates werenot stable in the presence of a large excess of high molecular weightsalmon sperm DNA used as model for the background DNA that wouldnormally be present in a diagnostic system. Longer exposure of thecolloids to the thiol-oligonucleotides produced oligonucleotide-colloidconjugates that were stable to salmon sperm DNA, but the resultingconjugates failed to hybridize satisfactorily. Further experimentationled to the following procedure for attaching thiol-oligonucleotides ofany length to gold colloids so that the conjugates are stable to highmolecular weight DNA and hybridize satisfactorily.

[0276] A 1 mL solution of the gold colloids (17nM) in water was mixedwith excess (3.68 μM) thiol-oligonucleotide (28 bases in length) inwater, and the mixture was allowed to stand for 12-24 hours at roomtemperature. Then, 100 μL of a 0.1 M sodium hydrogen phosphate buffer,pH 7.0, and 100 μL of 1.0 M NaCl were premixed and added. After 10minutes, 10 μL of 1% aqueous NaN₃ were added, and the mixture wasallowed to stand for an additional 40 hours. This “aging” step wasdesigned to increase the surface coverage by the thiol-oligonucleotidesand to displace oligonucleotide bases from the gold surface. Somewhatcleaner, better defined red spots in subsequent assays were obtained ifthe solution was frozen in a dry-ice bath after the 40-hour incubationand then thawed at room temperature. Either way, the solution was nextcentrifuged at 14,000 rpm in an Eppendorf Centrifuge 5414 for about 15minutes to give a very pale pink supernatant containing most of theoligonucleotide (as indicated by the absorbance at 260 nm) along with7-10% of the colloidal gold (as indicated by the absorbance at 520 nm),and a compact, dark, gelatinous residue at the bottom of the tube. Thesupernatant was removed, and the residue was resuspended in about 200 μLof buffer (10 mM phosphate, 0.1 M NaCl) and recentrifuged. After removalof the supernatant solution, the residue was taken up in 1.0 mL ofbuffer (10 mM phosphate, 0.1 M NaCl) and 10 μL of a 1% aqueous solutionof NaN₃. Dissolution was assisted by drawing the solution into, andexpelling it from, a pipette several times. The resulting red mastersolution was stable (. e., remained red and did not aggregate) onstanding for months at room temperature, on spotting on silicathin-layer chromatography (TLC) plates (see Example 4), and on additionto 2 M NaCl, 10 mM MgCl₂, or solutions containing high concentrations ofsalmon sperm DNA.

Example 4 Acceleration of Hybridization of Nanoparticle-oligonucleotideConjugates

[0277] The oligonucleotide-gold colloid conjugates I and II illustratedin FIG. 11 were prepared as described in Example 3. The hybridization ofthese two conjugates was extremely slow. In particular, mixing samplesof conjugates I and II in aqueous 0.1 M NaCl or in 10 mM MgCl₂ plus 0.1M NaCl and allowing the mixture to stand at room temperature for a dayproduced little or no color change.

[0278] Two ways were found to improve hybridization. First, fasterresults were obtained by freezing the mixture of conjugates I and II(each 15 nM contained in a solution of 0.1 M NaCl) in a dryice-isopropyl alcohol bath for 5 minutes and then thawing the mixture atroom temperature. The thawed solution exhibited a bluish color. When 1μL of the solution was spotted on a standard C-18 TLC silica plate(Alltech Associates), a strong blue color was seen immediately. Thehybridization and consequent color change caused by the freeze-thawingprocedure were reversible. On heating the hybridized solution to 80° C.,the solution turned red and produced a pink spot on a TLC plate.Subsequent freezing and thawing returned the system to the (blue)hybridized state (both solution and spot on a C-18 TLC plate). In asimilar experiment in which the solution was not refrozen, the spotobtained on the C-18 TLC plate was pink.

[0279] A second way to obtain faster results is to warm the conjugatesand target. For instance, in another experiment, oligonucleotide-goldcolloid conjugates and an oligonucleotide target sequence in a 0.1 MNaCl solution were warmed rapidly to 65° C. and allowed to cool to roomtemperature over a period of 20 minutes. On spotting on a C-18 silicaplate and drying, a blue spot indicative of hybridization was obtained.In contrast, incubation of the conjugates and target at room temperaturefor an hour in 0.1 M NaCl solution did not produce a blue colorindicative of hybridization. Hybridization is more rapid in 0.3 M NaCl.

Example 5 Assays Using Nanoparticle-oligonucleotide Conjugates

[0280] The oligonucleotide-gold colloid conjugates 1 and 2 illustratedin FIGS. 1 2A-F were prepared as described in Example 3, and theoligonucleotide target 3 illustrated in FIG. 12A was prepared asdescribed in Example 2. Mismatched and deletion targets 4, 5, 6, and 7were purchased from the Northwestern University Biotechnology Facility,Chicago, Ill. These oligonucleotides were synthesized on a 40 nmol scaleand purified on an reverse phase C18 cartridge (OPC). Their purity wasdetermined by performing ion exchange HPLC.

[0281] Selective hybridization was achieved by heating rapidly and thencooling rapidly to the stringent temperature. For example, hybridizationwas carried out in 100 μL of 0.1 M NaCl plus 5 mM MgCl₂ containing 15 nMof each oligonucleotide-colloid conjugate 1 and 2, and 3 nanomoles oftarget oligonucleotide 3, 4, 5, 6, or 7, heating to 74° C., cooling tothe temperatures indicated in Table 1 below, and incubating the mixtureat this temperature for 10 minutes. A 3 μL sample of each reactionmixture was then spotted on a C-18 T LC silica plate. On drying (5minutes), a strong blue color appeared if hybridization had taken place.

[0282] The results are presented in Table 1 below. Pink spots signify anegative test (i.e., that the nanoparticles were not brought together byhybridization), and blue spots signify a positive test (i.e., that thenanoparticles were brought into proximity due to hybridization involvingboth of the oligonucleotide-colloid conjugates).

We claim:
 1. A method of detecting a nucleic acid having at least twoportions comprising: providing a type of nanoparticles havingoligonucleotides attached thereto, the oligonucleotides on eachnanoparticle having a sequence complementary to the sequence of at leasttwo portions of the nucleic acid; contacting the nucleic acid and thenanoparticles under conditions effective to allow hybridization of theoligonucleotides on the nanoparticles with the two or more portions ofthe nucleic acid; and observing a detectable change brought about byhybridization of the oligonucleotides on the nanoparticles with thenucleic acid.
 2. A method of detecting nucleic acid having at least twoportions comprising: contacting the nucleic acid with at least two typesof nanoparticles having oligonucleotides attached thereto, theoligonucleotides on the first type of nanoparticles having a sequencecomplementary to a first portion of the sequence of the nucleic acid,the oligonucleotides on the second type of nanoparticles having asequence complementary to a second portion of the sequence of thenucleic acid, the contacting taking place under conditions effective toallow hybridization of the oligonucleotides on the nanoparticles withthe nucleic acid; and observing a detectable change brought about byhybridization of the oligonucleotides on the nanoparticles with thenucleic acid.
 3. The method of claim 2 wherein the contacting conditionsinclude freezing and thawing.
 4. The method of claim 2 wherein thecontacting conditions include heating.
 5. The method of claim 2 whereinthe detectable change is observed on a solid surface.
 6. The method ofclaim 2 wherein the detectable change is a color change observable withthe naked eye.
 7. The method of claim 6 wherein the color change isobserved on a solid surface.
 8. The method of claim 2 wherein thenanoparticles are made of gold.
 9. The method of claim 2 wherein theoligonucleotides attached to the nanoparticles are labeled on their endsnot attached to the nanoparticles with molecules that produce adetectable change upon hybridization of the oligonucleotides on thenanoparticles with the nucleic acid.
 10. The method of claim 9 whereinthe nanoparticles are metallic or semiconductor nanoparticles and theoligonucleotides attached to the nanoparticles are labeled withfluorescent molecules.
 11. The method of claim 2 wherein: the nucleicacid has a third portion located between the first and second portions,and the sequences of the oligonucleotides on the nanoparticles do notinclude sequences complementary to this third portion of the nucleicacid; and the nucleic acid is further contacted with a filleroligonucleotide having a sequence complementary to this third portion ofthe nucleic acid, the contacting taking place under conditions effectiveto allow hybridization of the filler oligonucleotide with the nucleicacid.
 12. The method of claim 2 wherein the nucleic acid is viral RNA orDNA.
 13. The method of claim 2 wherein the nucleic acid is a geneassociated with a disease.
 14. The method of claim 2 wherein the nucleicacid is a bacterial DNA.
 15. The method of claim 2 wherein the nucleicacid is a fungal DNA.
 16. The method of claim 2 wherein the nucleic acidis a synthetic DNA, a synthetic RNA, a structurally-modified natural orsynthetic RNA, or a structurally-modified natural or synthetic DNA. 17.The method of claim 2 wherein the nucleic acid is from a biologicalsource.
 18. The method of claim 2 wherein the nucleic acid is a productof a polymerase chain reaction amplification.
 19. The method of claim 2wherein the nucleic acid is contacted with the first and second types ofnanoparticles simultaneously.
 20. The method of claim 2 wherein thenucleic acid is contacted and hybridized with the oligonucleotides onthe first type of nanoparticles before being contacted with the secondtype of nanoparticles.
 21. The method of claim 20 wherein the first typeof nanoparticles is attached to a substrate.
 22. The method of claim 2wherein the nucleic acid is double-stranded and hybridization with theoligonucleotides on the nanoparticles results in the production of atriple-stranded complex.
 23. A method of detecting nucleic acid havingat least two portions comprising: providing a substrate having a firsttype of nanoparticles attached thereto, the nanoparticles havingoligonucleotides attached thereto, the oligonucleotides having asequence complementary to a first portion of the sequence of a nucleicacid to be detected; contacting said nucleic acid with the nanoparticlesattached to the substrate under conditions effective to allowhybridization of the oligonucleotides on the nanoparticles with saidnucleic acid; providing a second type of nanoparticles havingoligonucleotides attached thereto, the oligonucleotides having asequence complementary to one or more other portions of the sequence ofsaid nucleic acid; contacting said nucleic acid bound to the substratewith the second type of nanoparticles under conditions effective toallow hybridization of the oligonucleotides on the second type ofnanoparticles with said nucleic acid; and observing a detectable change.24. The method of claim 23 wherein the substrate has a plurality oftypes of nanoparticles attached to it in an array to allow for thedetection of multiple portions of a single nucleic acid, the detectionof multiple different nucleic acids, or both.
 25. A method of detectingnucleic acid having at least two portions comprising: providing asubstrate having a first type of nanoparticles attached thereto, thenanoparticles having oligonucleotides attached thereto, theoligonucleotides having a sequence complementary to a first portion ofthe sequence of a nucleic acid to be detected; contacting said nucleicacid with the nanoparticles attached to the substrate under conditionseffective to allow hybridization of the oligonucleotides on thenanoparticles with said nucleic acid; providing a second type ofnanoparticles having oligonucleotides attached thereto, theoligonucleotides having a sequence complementary to one or more otherportions of the sequence of said nucleic acid; contacting said nucleicacid bound to the substrate with the second type of nanoparticles underconditions effective to allow hybridization of the oligonucleotides onthe second type of nanoparticles with said nucleic acid; providing abinding oligonucleotide having a selected sequence having at least twoportions, the first portion being complementary to at least a portion ofthe sequence of the oligonucleotides on the second type ofnanoparticles; contacting the binding oligonucleotide with the secondtype of nanoparticles bound to the substrate under conditions effectiveto allow hybridization of the binding oligonucleotide to theoligonucleotides on the nanoparticles; providing a third type ofnanoparticles having oligonucleotides attached thereto, theoligonucleotides having a sequence complementary to the sequence of asecond portion of the binding oligonucleotide; contacting the third typeof nanoparticles with the binding oligonucleotide bound to the substrateunder conditions effective to allow hybridization of the bindingoligonucleotide to the oligonucleotides on the nanoparticles; andobserving a detectable change.
 26. The method of claim 25 wherein thesubstrate has a plurality of types of nanoparticles attached to it in anarray to allow for the detection of multiple portions of a singlenucleic acid, the detection of multiple different nucleic acids, orboth.
 27. A method of detecting nucleic acid having at least twoportions comprising: contacting a nucleic acid to be detected with asubstrate having oligonucleotides attached thereto, the oligonucleotideshaving a sequence complementary to a first portion of the sequence ofsaid nucleic acid, the contacting taking place under conditionseffective to allow hybridization of the oligonucleotides on thesubstrate with said nucleic acid; contacting said nucleic acid bound tothe substrate with a first type of nanoparticles having one or moretypes of oligonucleotides attached thereto, at least one of the types ofoligonucleotides having a sequence complementary to a second portion ofthe sequence of said nucleic acid, the contacting taking place underconditions effective to allow hybridization of the oligonucleotides onthe nanoparticles with said nucleic acid; contacting the first type ofnanoparticles bound to the substrate with a second type of nanoparticleshaving oligonucleotides attached thereto, the oligonucleotides on thesecond type of nanoparticles having a sequence complementary to at leasta portion of the sequence of one of the types of oligonucleotides on thefirst type of nanoparticles, the contacting taking place underconditions effective to allow hybridization of the oligonucleotides onthe first and second types of nanoparticles; and observing a detectablechange.
 28. The method of claim 27 wherein the first type ofnanoparticles has only one type of oligonucleotides attached thereto,the oligonucleotides having a sequence complementary to the secondportion of the sequence of said nucleic acid and to at least a portionof the sequence of the oligonucleotides on the second type ofnanoparticles.
 29. The method of claim 28 further comprising contactingthe second type of nanoparticles bound to the substrate with the firsttype of nanoparticles, the contacting taking place under conditionseffective to allow hybridization of the oligonucleotides on the firstand second types of nanoparticles.
 30. The method of claim 27 whereinthe first type of nanoparticles has at least two types ofoligonucleotides attached thereto, the first type of oligonucleotideshaving a sequence complementary to the second portion of the sequence ofsaid nucleic acid, and the second type of oligonucleotides having asequence complementary to the sequence of at least a portion of theoligonucleotides on the second type of nanoparticles.
 31. The method ofclaim 30 further comprising contacting the second type of nanoparticlesbound to the substrate with the first type of nanoparticles, thecontacting taking place under conditions effective to allowhybridization of the oligonucleotides on the first and second types ofnanoparticles.
 32. The method of claim 27 wherein the substrate has aplurality of types of oligonucleotides attached to it in an array toallow for the detection of multiple portions of a single nucleic acid,the detection of multiple different nucleic acids, or both.
 33. Themethod of any one of claims 23-32 wherein the substrate is a transparentsubstrate or an opaque white substrate.
 34. The method of claim 33wherein the detectable change is the formation of dark areas on thesubstrate.
 35. The method of any one of claims 23-32 wherein thenanoparticles are made of gold.
 36. The method of any one of claims23-32 wherein the substrate is contacted with silver stain to producethe detectable change.
 37. The method of any one of claims 23-32 whereinthe detectable change is observed with an optical scanner.
 38. A methodof detecting nucleic acid having at least two portions comprising:contacting a nucleic acid to be detected with a substrate havingoligonucleotides attached thereto, the oligonucleotides having asequence complementary to a first portion of the sequence of saidnucleic acid, the contacting taking place under conditions effective toallow hybridization of the oligonucleotides on the substrate with saidnucleic acid; contacting said nucleic acid bound to the substrate with atype of nanoparticles having oligonucleotides attached thereto, theoligonucleotides having a sequence complementary to a second portion ofthe sequence of said nucleic acid, the contacting taking place underconditions effective to allow hybridization of the oligonucleotides onthe nanoparticles with said nucleic acid; contacting the substrate withsilver stain to produce a detectable change; and observing thedetectable change.
 39. The method of claim 38 wherein the nanoparticlesare made of a noble metal.
 40. The method of claim 39 wherein thenanoparticles are made of gold or silver.
 41. The method of claim 38wherein the substrate has a plurality of types of oligonucleotidesattached to it in an array to allow for the detection of multipleportions of a single nucleic acid, the detection of multiple differentnucleic acids, or both.
 42. The method of any one of claims 38-41wherein the detectable change is observed with an optical scanner.
 43. Amethod of detecting nucleic acid having at least two portionscomprising: contacting a nucleic acid to be detected with a substratehaving oligonucleotides attached thereto, the oligonucleotides having asequence complementary to a first portion of the sequence of saidnucleic acid, the contacting taking place under conditions effective toallow hybridization of the oligonucleotides on the substrate with saidnucleic acid; contacting said nucleic acid bound to the substrate withliposomes having oligonucleotides attached thereto, the oligonucleotideshaving a sequence complementary to a portion of the sequence of saidnucleic acid, the contacting taking place under conditions effective toallow hybridization of the oligonucleotides on the liposomes with saidnucleic acid; contacting the liposomes bound to the substrate with afirst type of nanoparticles having at least a first typeoligonucleotides attached thereto, the first type of oligonucleotideshaving a hydrophobic group attached to the end not attached to thenanoparticles, the contacting taking place under conditions effective toallow attachment of the oligonucleotides on the nanoparticles to theliposomes as a result of hydrophobic interactions; and observing adetectable change.
 44. A method of detecting nucleic acid having atleast two portions comprising contacting a nucleic acid to be detectedwith a substrate having oligonucleotides attached thereto, theoligonucleotides having a sequence complementary to a first portion ofthe sequence of said nucleic acid, the contacting taking place underconditions effective to allow hybridization of the oligonucleotides onthe substrate with said nucleic acid; contacting said nucleic acid boundto the substrate with liposomes having oligonucleotides attachedthereto, the oligonucleotides having a sequence complementary to aportion of the sequence of said nucleic acid, the contacting takingplace under conditions effective to allow hybridization of theoligonucleotides on the liposomes with said nucleic acid; contacting theliposomes bound to the substrate with a first type of nanoparticleshaving at least a first type oligonucleotides attached thereto, thefirst type of oligonucleotides having a hydrophobic group attached tothe end not attached to the nanoparticles, the contacting taking placeunder conditions effective to allow attachment of the oligonucleotideson the nanoparticles to the liposomes as a result of hydrophobicinteractions; contacting the first type of nanoparticles bound to theliposomes with a second type of nanoparticles having oligonucleotidesattached thereto, the first type of nanoparticles having a second typeof oligonucleotides attached thereto which have a sequence complementaryto at least a portion of the sequence of the oligonucleotides on thesecond type of nanoparticles, the oligonucleotides on the second type ofnanoparticles having a sequence complementary to at least a portion ofthe sequence of the second type of oligonucleotides on the first type ofnanoparticles, the contacting taking place under conditions effective toallow hybridization of the oligonucleotides on the first and secondtypes of nanoparticles; and observing a detectable change.
 45. Themethod of claim 43 or 44 wherein the substrate has a plurality of typesof oligonucleotides attached to it in an array to allow for thedetection of multiple portions of a single nucleic acid, the detectionof multiple different nucleic acids, or both.
 46. The method of claim 43or 44 wherein the nanoparticles are made of gold.
 47. The method ofclaim 43 or 44 wherein the substrate is contacted with silver stain toproduce the detectable change.
 48. The method of any one of claims 43 or44 wherein the detectable change is observed with an optical scanner.49. A method of detecting nucleic acid having at least two portionscomprising: providing a substrate having a first type of nanoparticlesattached thereto, the nanoparticles having oligonucleotides attachedthereto, the oligonucleotides having a sequence complementary to a firstportion of the sequence of a nucleic acid to be detected; contactingsaid nucleic acid with the nanoparticles attached to the substrate underconditions effective to allow hybridization of the oligonucleotides onthe nanoparticles with said nucleic acid; providing an aggregate probecomprising at least two types of nanoparticles having oligonucleotidesattached thereto, the nanoparticles of the aggregate probe being boundto each other as a result of the hybridization of some of theoligonucleotides attached to them, at least one of the types ofnanoparticles of the aggregate probe having oligonucleotides attachedthereto which have a sequence complementary to a second portion of thesequence of said nucleic acid; contacting said nucleic acid bound to thesubstrate with the aggregate probe under conditions effective to allowhybridization of the oligonucleotides on the aggregate probe with saidnucleic acid; and observing a detectable change.
 50. The method of claim49 wherein the substrate has a plurality of types of nanoparticlesattached to it in an array to allow for the detection of multipleportions of a single nucleic acid, the detection of multiple differentnucleic acids, or both.
 51. A method of detecting nucleic acid having atleast two portions comprising: providing a substrate havingoligonucleotides attached thereto, the oligonucleotides having asequence complementary to a first portion of the sequence of a nucleicacid to be detected; providing an aggregate probe comprising at leasttwo types of nanoparticles having oligonucleotides attached thereto, thenanoparticles of the aggregate probe being bound to each other as aresult of the hybridization of some of the oligonucleotides attached tothem, at least one of the types of nanoparticles of the aggregate probehaving oligonucleotides attached thereto which have a sequencecomplementary to a second portion of the sequence of said nucleic acid;contacting said nucleic acid, the substrate and the aggregate probeunder conditions effective to allow hybridization of said nucleic acidwith the oligonucleotides on the aggregate probe and with theoligonucleotides on the substrate; and observing a detectable change.52. The method of claim 51 wherein said nucleic acid is contacted withthe substrate so that said nucleic acid hybridizes with theoligonucleotides on the substrate, and said nucleic acid bound to thesubstrate is then contacted with the aggregate probe so that saidnucleic acid hybridizes with the oligonucleotides on the aggregateprobe.
 53. The method of claim 51 wherein said nucleic acid is contactedwith the aggregate probe so that said nucleic acid hybridizes with theoligonucleotides on the aggregate probe, and said nucleic acid bound tothe aggregate probe is then contacted with the substrate so that saidnucleic acid hybridizes with the oligonucleotides on the substrate. 54.The method of claim 51 wherein said nucleic acid is contactedsimultaneously with the aggregate probe and the substrate.
 55. Themethod of claim 51 wherein the substrate has a plurality of types ofoligonucleotides attached to it in an array to allow for the detectionof multiple portions of a single nucleic acid, the detection of multipledifferent nucleic acids, or both.
 56. A method of detecting nucleic acidhaving at least two portions comprising: providing a substrate havingoligonucleotides attached thereto; providing an aggregate probecomprising at least two types of nanoparticles having oligonucleotidesattached thereto, the nanoparticles of the aggregate probe being boundto each other as a result of the hybridization of some of theoligonucleotides attached to them, at least one of the types ofnanoparticles of the aggregate probe having oligonucleotides attachedthereto which have a sequence complementary to a first portion of thesequence of a nucleic acid to be detected; providing a type ofnanoparticles having at least two types of oligonucleotides attachedthereto, the first type of oligonucleotides having a sequencecomplementary to a second portion of the sequence of said nucleic acid,the second type of oligonucleotides having a sequence complementary toat least a portion of the sequence of the oligonucleotides attached tothe substrate; contacting said nucleic acid, the aggregate probe, thenanoparticles and the substrate, the contacting taking place underconditions effective to allow hybridization of said nucleic acid withthe oligonucleotides on the aggregate probe and on the nanoparticles andhybridization of the oligonucleotides on the nanoparticles with theoligonucleotides on the substrate; and observing a detectable change.57. The method of claim 56 wherein said nucleic acid is contacted withthe aggregate probe and the nanoparticles so that said nucleic acidhybridizes with the oligonucleotides on the aggregate probe and with theoligonucleotides on the nanoparticles, and said nucleic acid bound tothe aggregate probe and nanoparticles is then contacted with thesubstrate so that the oligonucleotides on the nanoparticles hybridizewith the oligonucleotides on the substrate.
 58. The method of claim 56wherein said nucleic acid is contacted with the aggregate probe so thatsaid nucleic acid hybridizes with the oligonucleotides on the aggregateprobe, said nucleic acid bound to the aggregate probe is then contactedwith the nanoparticles so that said nucleic acid hybridizes with theoligonucleotides on the nanoparticles, and said nucleic acid bound tothe aggregate probe and nanoparticles is then contacted with thesubstrate so that the oligonucleotides on the nanoparticles hybridizewith the oligonucleotides on the substrate.
 59. The method of claim 56wherein said nucleic acid is contacted with the aggregate probe so thatsaid nucleic acid hybridizes with the oligonucleotides on the aggregateprobe, the nanoparticles are contacted with the substrate so that theoligonucleotides on the nanoparticles hybridize with theoligonucleotides on the substrate, and said nucleic acid bound to theaggregate probe is then contacted with the nanoparticles bound to thesubstrate so that said nucleic acid hybridizes with the oligonucleotideson the nanoparticles.
 60. The method of claim 56 wherein the substratehas the olligonucleotides attached to it in an array to allow for thedetection of multiple portions of a single nucleic acid, the detectionof multiple different nucleic acids, or both.
 61. The method of any oneof claims 49-60 wherein the substrate is a transparent substrate or anopaque white substrate.
 62. The method of claim 61 wherein thedetectable change is the formation of dark areas on the substrate. 63.The method of any one of claims 49-60 wherein the nanoparticles in theaggregate probe are made of gold.
 64. The method of any one of claims49-60 wherein the substrate is contacted with a silver stain to producethe detectable change.
 65. The method of any one of claims 49-60 whereinthe detectable change is observed with an optical scanner.
 66. A methodof detecting nucleic acid having at least two portions comprising:contacting a nucleic acid to be detected with a substrate havingoligonucleotides attached thereto, the oligonucleotides having asequence complementary to a first portion of the sequence of saidnucleic acid, the contacting taking place under conditions effective toallow hybridization of the oligonucleotides on the substrate with saidnucleic acid; contacting said nucleic acid bound to the substrate withliposomes having oligonucleotides attached thereto, the oligonucleotideshaving a sequence complementary to a portion of the sequence of saidnucleic acid, the contacting taking place under conditions effective toallow hybridization of the oligonucleotides on the liposomes with saidnucleic acid; providing an aggregate probe comprising at least two typesof nanoparticles having oligonucleotides attached thereto, thenanoparticles of the aggregate probe being bound to each other as aresult of the hybridization of some of the oligonucleotides attached tothem, at least one of the types of nanoparticles of the aggregate probehaving oligonucleotides attached thereto which have a hydrophobic groupattached to the end not attached to the nanoparticles; contacting theliposomes bound to the substrate with the aggregate probe underconditions effective to allow attachment of the oligonucleotides on theaggregate probe to the liposomes as a result of hydrophobicinteractions; and observing a detectable change.
 67. The method of claim66 wherein the nanoparticles in the aggregate probe are made of gold.68. The method of claim 66 wherein the substrate is contacted with asilver stain to produce the detectable change.
 69. The method of claim66 wherein the substrate has a plurality of types of oligonucleotidesattached to it in an array to allow for the detection of multipleportions of a single nucleic acid, the detection of multiple differentnucleic acids, or both.
 70. A method of detecting nucleic acid having atleast two portions comprising: providing a substrate havingoligonucleotides attached thereto, the oligonucleotides having asequence complementary to a first portion of the sequence of a nucleicacid to be detected; providing a core probe comprising at least twotypes of nanoparticles, each type of nanoparticles havingoligonucleotides attached thereto which are complementary to theoligonucleotides on at least one of the other types of nanoparticles,the nanoparticles of the aggregate probe being bound to each other as aresult of the hybridization of the oligonucleotides attached to them;providing a type of nanoparticles having two types of oligonucleotidesattached thereto, the first type of oligonucleotides having a sequencecomplementary to a second portion of the sequence of said nucleic acid,the second type of oligonucleotides having a sequence complementary to aportion of the sequence of the oligonucleotides attached to at least oneof the types of nanoparticles of the core probe; contacting said nucleicacid, the nanoparticles, the substrate and the core probe underconditions effective to allow hybridization of said nucleic acid withthe oligonucleotides on the nanoparticles and with the oligonucleotideson the substrate and to allow hybridization of the oligonucleotides onthe nanoparticles with the oligonucleotides on the core probe; andobserving a detectable change.
 71. The method of claim 70 wherein saidnucleic acid is contacted with the substrate so that said nucleic acidhybridizes with the oligonucleotides on the substrate, and said nucleicacid bound to the substrate is then contacted with the nanoparticles sothat said nucleic acid hybridizes with the oligonucleotides on thenanoparticles, and the nanoparticles bound to said nucleic acid arecontacted with the core probe so that the oligonucleotides on the coreprobe hybridize with the oligonucleotides on the nanoparticles.
 72. Themethod of claim 70 wherein said nucleic acid is contacted with thenanoparticles so that said nucleic acid hybridizes with theoligonucleotides on the nanoparticles, said nucleic acid bound to thenanoparticles is then contacted with the substrate so that said nucleicacid hybridizes with the oligonucleotides on the substrate, and thenanoparticles bound to said nucleic acid are contacted with the coreprobe so that the oligonucleotides on the core probe hybridize with theoligonucleotides on the nanoparticles.
 73. A method of detecting nucleicacid having at least two portions comprising: providing a substratehaving oligonucleotides attached thereto, the oligonucleotides having asequence complementary to a first portion of the sequence of a nucleicacid to be detected; providing a core probe comprising at least twotypes of nanoparticles, each type of nanoparticles havingoligonucleotides attached thereto which are complementary to theoligonucleotides on at least one other type of nanoparticles, thenanoparticles of the aggregate probe being bound to each other as aresult of the hybridization of the oligonucleotides attached to them;providing a type of linking oligonucleotides comprising a sequencecomplementary to a second portion of the sequence of said nucleic acidand a sequence complementary to a portion of the sequence of theoligonucleotides attached to at least one of the types of nanoparticlesof the core probe; contacting said nucleic acid, the linkingoligonucleotides, the substrate and the core probe under conditionseffective to allow hybridization of said nucleic acid with the linkingoligonucleotides and with the oligonucleotides on the substrate and toallow hybridization of the oligonucleotides on the linkingoligonucleotides with the oligonucleotides on the core probe; andobserving a detectable change.
 74. The method of any one of claims 70-73wherein the substrate has a plurality of types of oligonucleotidesattached to it in an array to allow for the detection of multipleportions of a single nucleic acid, the detection of multiple differentnucleic acids, or both.
 75. The method of any one of claims 70-73wherein the substrate is a transparent substrate or an opaque whitesubstrate.
 76. The method of claim 76 wherein the detectable change isthe formation of dark areas on the substrate.
 77. The method of any oneof claims 70-73 wherein the nanoparticles in the core probe are made ofgold.
 78. The method of any one of claims 70-73 wherein the substrate iscontacted with a silver stain to produce the detectable change.
 79. Themethod of any one of claims 70-73 wherein the detectable change isobserved with an optical scanner.
 80. A method of detecting a nucleicacid having at least two portions comprising: providing nanoparticleshaving oligonucleotides attached thereto; providing one or more types ofbinding oligonucleotides, each of the binding oligonucleotides havingtwo portions, the sequence of one portion being complementary to thesequence of one of the portions of the nucleic acid and the sequence ofthe other portion being complementary to the sequence of theoligonucleotides on the nanoparticles; contacting the nanoparticles andthe binding oligonucleotides under conditions effective to allowhybridization of the oligonucleotides on the nanoparticles with thebinding oligonucleotides; contacting the nucleic acid and the bindingoligonucleotides under conditions effective to allow hybridization ofthe binding oligonucleotides with the nucleic acid; and observing adetectable change.
 81. The method of claim 80 wherein the nanoparticlesare contacted with the binding oligonucleotides prior to being contactedwith the nucleic acid.
 82. A method of detecting a nucleic acid havingat least two portions comprising: providing nanoparticles havingoligonucleotides attached thereto; providing one or more bindingoligonucleotides, each of the binding oligonucleotides having twoportions, the sequence of one portion being complementary to thesequence of at least two portions of the nucleic acid and the sequenceof the other portion being complementary to the sequence of theoligonucleotides on the nanoparticles; contacting the nanoparticles andthe binding oligonucleotides under conditions effective to allowhybridization of the oligonucleotides on the nanoparticles with thebinding oligonucleotides; contacting the nucleic acid and the bindingoligonucleotides under conditions effective to allow hybridization ofthe binding oligonucleotides with the nucleic acid; and observing adetectable change.
 83. A method of detecting nucleic acid having atleast two portions comprising: contacting the nucleic acid with at leasttwo types of particles having oligonucleotides attached thereto, theoligonucleotides on the first type of particles having a sequencecomplementary to a first portion of the sequence of the nucleic acid andbeing labeled with an energy donor, the oligonucleotides on the secondtype of particles having a sequence complementary to a second portion ofthe sequence of the nucleic acid and being labeled with an energyacceptor, the contacting taking place under conditions effective toallow hybridization of the oligonucleotides on the particles with thenucleic acid; and observing a detectable change brought about byhybridization of the oligonucleotides on the particles with the nucleicacid.
 84. The method of claim 83 wherein the energy donor and acceptorare fluorescent molecules.
 85. A method of detecting nucleic acid havingat least two portions comprising: providing a type of microsphereshaving oligonucleotides attached thereto, the oligonucleotides having asequence complementary to a first portion of the sequence of the nucleicacid and being labeled with a fluorescent molecule; providing a type ofnanoparticles having oligonucleotides attached thereto, theoligonucleotides having a sequence complementary to a second portion ofthe sequence of the nucleic acid, nanoparticles being capable ofproducing a detectable change; contacting the nucleic acid with themicrospheres and the nanoparticles under conditions effective to allowhybridization of the oligonucleotides on the microspheres and on thenanoparticles with the nucleic acid; and observing a change influorescence, another detectable change produced by the nanoparticles,or both.
 86. The method of claim 85 wherein the detectable changeproduced by the nanoparticles is a change in color.
 87. The method ofclaim 85 wherein the microspheres are latex microspheres and thenanoparticles are gold nanoparticles, and changes in fluorescence, coloror both are observed.
 88. The method of claim 87 further comprisingplacing a portion of the mixture of the latex microspheres,nanoparticles and nucleic acid in an observation area located on amicroporous material, treating the microporous material so as to removeany unbound gold nanoparticles from the observation area, and thenobserving the changes in fluorescence, color, or both.
 89. A method ofdetecting nucleic acid having at least two portions comprising:providing a first type of metallic or semiconductor nanoparticles havingoligonucleotides attached thereto, the oligonucleotides having asequence complementary to a first portion of the sequence of the nucleicacid and being labeled with a fluorescent molecule; providing a secondtype of metallic or semiconductor nanoparticles having oligonucleotidesattached thereto, the oligonucleotides having a sequence complementaryto a second portion of the sequence of the nucleic acid and beinglabeled with a fluorescent molecule; contacting the nucleic acid withthe two types of nanoparticles under conditions effective to allowhybridization of the oligonucleotides on the two types of nanoparticleswith the nucleic acid; and observing changes in fluorescence.
 90. Themethod of claim 89 further comprising placing a portion of the mixtureof the nanoparticles and nucleic acid in an observation area located ona microporous material, treating the microporous material so as toremove any unbound nanoparticles from the observation area, and thenobserving the changes in fluorescence.
 91. A method of detecting nucleicacid having at least two portions comprising: providing a type ofparticle having oligonucleotides attached thereto, the oligonucleotideshaving a first portion and a second portion, both portions beingcomplementary to portions of the sequence of the nucleic acid; providinga type of probe oligonucleotides comprising a first portion and a secondportion, the first portion having a sequence complementary to the firstportion of the oligonucleotides attached to the particles and bothportions being complementary to portions of the sequence of the nucleicacid, the probe oligonucleotides further being labeled with a reportermolecule at one end; contacting the particle and the probeoligonucleotides under conditions effective to allow for hybridizationof the oligonucleotides on the particles with the probe oligonucleotidesto produce a satellite probe; then contacting the satellite probe withthe nucleic acid under conditions effective to provide for hybridizationof the nucleic acid with the probe oligonucleotides; removing theparticles; and detecting the reporter molecule.
 92. The method of claim91 wherein the particles are magnetic and the reporter molecule is afluorescent molecule.
 93. The method of claim 91 wherein the particlesare magnetic and the reporter molecule is a dye molecule.
 94. The methodof claim 91 wherein the particles are magnetic and the reporter moleculeis a redox-active molecule.
 95. A kit comprising at least one container,the container holding a composition comprising at least two types ofnanoparticles having oligonucleotides attached thereto, theoligonucleotides on the first type of nanoparticles having a sequencecomplementary to the sequence of a first portion of a nucleic acid, theoligonucleotides on the second type of nanoparticles having a sequencecomplementary to the sequence of a second portion of the nucleic acid.96. The kit of claim 95 wherein the composition in the container furthercomprises a filler oligonucleotide having a sequence complementary to athird portion of the nucleic acid, the third portion being locatedbetween the first and second portions.
 97. The kit of claim 95 whereinthe nanoparticles are made of gold.
 98. The kit of claim 95 furthercomprising a solid surface.
 99. A kit comprising at least twocontainers, the first container holding nanoparticles havingoligonucleotides attached thereto which have a sequence complementary tothe sequence of a first portion of a nucleic acid, and the secondcontainer holding nanoparticles having oligonucleotides attached theretowhich have a sequence complementary to the sequence of a second portionof the nucleic acid.
 100. The kit of claim 99 comprising a thirdcontainer holding oligonucleotides having a sequence complementary to athird portion of the nucleic acid, the third portion being locatedbetween the first and second portions.
 101. The kit of claim 99 whereinthe nanoparticles are made of gold.
 102. The kit of claim 99 furthercomprising a solid surface.
 103. A kit comprising at least twocontainers, the first container holding nanoparticles havingoligonucleotides attached thereto which have a sequence complementary tothe sequence of a first portion of a binding oligonucleotide, and thesecond container holding one or more types of binding oligonucleotides,each of which has a sequence comprising at least two portions, the firstportion being complementary to the sequence of the oligonucleotides onthe nanoparticles and the second portion being complementary to thesequence of a portion of a nucleic acid.
 104. The kit of claim 103 whichcomprises additional containers, each holding an additional bindingoligonucleotide, each additional binding oligonucleotide having asequence comprising at least two portions, the first portion beingcomplementary to the sequence of the oligonucleotides on thenanoparticles and the second portion being complementary to the sequenceof another portion of the nucleic acid.
 105. The kit of claim 103wherein the nanoparticles are made of gold.
 106. The kit of claim 103further comprising a solid surface.
 107. A kit comprising: a containerholding one type of nanoparticles having oligonucleotides attachedthereto and one or more types of binding oligonucleotides, each of thetypes of binding oligonucleotides having a sequence comprising at leasttwo portions, the first portion being complementary to the sequence ofthe oligonucleotides on the nanoparticles, whereby the bindingoligonucleotides are hybridized to the oligonucleotides on thenanoparticles, and the second portion being complementary to thesequence of one or more portions of a nucleic acid.
 108. A kitcomprising at least one container, the container holding metallic orsemiconductor nanoparticles having oligonucleotides attached thereto,the oligonucleotides having a sequence complementary to a portion of anucleic acid and having fluorescent molecules attached to the ends ofthe oligonucleotides not attached to the nanoparticles.
 109. A kitcomprising: a substrate, the substrate having attached theretonanoparticles, the nanoparticles having oligonucleotides attachedthereto which have a sequence complementary to the sequence of a firstportion of a nucleic acid; and a first container holding nanoparticleshaving oligonucleotides attached thereto which have a sequencecomplementary to the sequence of a second portion of the nucleic acid.110. The kit of claim 109 further comprising: a second container holdinga binding oligonucleotide having a selected sequence having at least twoportions, the first portion being complementary to at least a portion ofthe sequence of the oligonucleotides on the nanoparticles in the firstcontainer; and a third container holding nanoparticles havingoligonucleotides attached thereto, the oligonucleotides having asequence complementary to the sequence of a second portion of thebinding oligonucleotide.
 111. A kit comprising at least threecontainers: the first container holding nanoparticles; the secondcontainer holding a first oligonucleotide having a sequencecomplementary to the sequence of a first portion of a nucleic acid; andthe third container holding a second oligonucleotide having a sequencecomplementary to the sequence of a second portion of the nucleic acid.112. The kit of claim 111 further comprising a fourth container holdinga third oligonucleotide having a sequence complementary to the sequenceof a third portion of the nucleic acid, the third portion being locatedbetween the first and second portions.
 113. The kit of claim 111 furthercomprising a substrate.
 114. The kit of claim 113 further comprising: afourth container holding a binding oligonucleotide having a selectedsequence having at least two portions, the first portion beingcomplementary to at least a portion of the sequence of the secondoligonucleotide; and a fifth container holding an oligonucleotide havinga sequence complementary to the sequence of a second portion of thebinding oligonucleotide.
 115. The kit of claim 111 wherein theoligonucleotides, nanoparticles, or both bear functional groups forattachment of the oligonucleotides to the nanoparticles.
 116. The kit ofclaim 113 wherein the substrate, nanoparticles, or both bear functionalgroups for attachment of the nanoparticles to the substrate.
 117. Thekit of claim 113 wherein the substrate has nanoparticles attached to it.118. The kit of claim 111 wherein the nanoparticles are made of gold.119. A kit comprising: a substrate having oligonucleotides attachedthereto which have a sequence complementary to the sequence of a firstportion of a nucleic acid; a first container holding nanoparticleshaving oligonucleotides attached thereto, some of which have a sequencecomplementary to the sequence of a second portion of the nucleic acid;and a second container holding nanoparticles having oligonucleotidesattached thereto which have a sequence complementary to at least aportion of the sequence of the oligonucleotides attached to thenanoparticles in the first container.
 120. A kit comprising: asubstrate; a first container holding nanoparticles; a second containerholding a first oligonucleotide having a sequence complementary to thesequence of a first portion of a nucleic acid; a third container holdinga second oligonucleotide having a sequence complementary to the sequenceof a second portion of the nucleic acid; and a fourth container holdinga third oligonucleotide having a sequence complementary to at least aportion of the sequence of the second oligonucleotide.
 121. The kit ofclaim 120 wherein the oligonucleotides, nanoparticles, substrate or allbear functional groups for attachment of the oligonucleotides to thenanoparticles or for attachment of the oligonucleotides to thesubstrate.
 122. The kit of claim 120 wherein the nanoparticles are madeof gold.
 123. A kit comprising: a substrate having oligonucleotidesattached thereto which have a sequence complementary to the sequence ofa first portion of a nucleic acid; a first container holding liposomeshaving oligonucleotides attached thereto which have a sequencecomplementary to the sequence of a second portion of the nucleic acid;and a second container holding nanoparticles having at least a firsttype of oligonucleotides attached thereto, the first type ofoligonucleotides having a hydrophobic group attached to the end notattached to the nanoparticles.
 124. The kit of claim 123 wherein: thenanoparticles in the second container have a second type ofoligonucleotides attached thereto, the second type of oligonucleotideshaving a sequence complementary to the sequence of the oligonucleotideson a second type of nanoparticles; and the kit further comprises: athird container holding a second type of nanoparticles havingoligonucleotides attached thereto, the oligonucleotides having asequence complementary to at least a portion of the sequence of thesecond type of oligonucleotides on the first type of nanoparticles. 125.A kit comprising: a substrate, the substrate having attached theretonanoparticles, the nanoparticles having oligonucleotides attachedthereto which have a sequence complementary to the sequence of a firstportion of a nucleic acid; and a first container holding an aggregateprobe comprising at least two types of nanoparticles havingoligonucleotides attached thereto, the nanoparticles of the aggregateprobe being bound to each other as a result of the hybridization of someof the oligonucleotides attached to them, at least one of the types ofnanoparticles of the aggregate probe having oligonucleotides attachedthereto which have a sequence complementary to a second portion of thesequence of the nucleic acid.
 126. A kit comprising: a substrate, thesubstrate having oligonucleotides attached thereto, the oligonucleotideshaving a sequence complementary to the sequence of a first portion of anucleic acid; and a first container holding an aggregate probecomprising at least two types of nanoparticles having oligonucleotidesattached thereto, the nanoparticles of the aggregate probe being boundto each other as a result of the hybridization of some of theoligonucleotides attached to them, at least one of the types ofnanoparticles of the aggregate probe having oligonucleotides attachedthereto which have a sequence complementary to a second portion of thesequence of the nucleic acid.
 127. The kit of claim 126 wherein thesubstrate has a plurality of types of oligonucleotides attached to it inan array to allow for the detection of multiple portions of a singlenucleic acid, the detection of multiple different nucleic acids, orboth.
 128. A kit comprising: a substrate having oligonucleotidesattached thereto; a first container holding an aggregate probecomprising at least two types of nanoparticles having oligonucleotidesattached thereto, the nanoparticles of the aggregate probe being boundto each other as a result of the hybridization of some of theoligonucleotides attached to them, at least one of the types ofnanoparticles of the aggregate probe having oligonucleotides attachedthereto which have a sequence complementary to a first portion of thesequence of the nucleic acid; and a second container holdingnanoparticles having at least two types of oligonucleotides attachedthereto, the first type of oligonucleotides having a sequencecomplementary to a second portion of the sequence of the nucleic acid,and the second type of oligonucleotides having a sequence complementaryto at least a portion of the sequence of the oligonucleotides attachedto the substrate.
 129. A kit comprising: a substrate, the substratehaving oligonucleotides attached thereto, the oligonucleotides having asequence complementary to the sequence of a first portion of a nucleicacid; a first container holding liposomes having oligonucleotidesattached thereto which have a sequence complementary to the sequence ofa second portion of the nucleic acid; and a second container holding anaggregate probe comprising at least two types of nanoparticles havingoligonucleotides attached thereto, the nanoparticles of the aggregateprobe being bound to each other as a result of the hybridization of someof the oligonucleotides attached to them, at least one of the types ofnanoparticles of the aggregate probe having oligonucleotides attachedthereto which have a hydrophobic group attached to the end not attachedto the nanoparticles.
 130. The kit of any one of claims 125-129 whereinthe substrate is a transparent substrate or an opaque white substrate.131. The kit of any one of claims 125-129 wherein the nanoparticles ofthe aggregate probe are made of gold.
 132. A kit comprising at leastthree containers: the first container holding nanoparticles; the secondcontainer holding a first oligonucleotide having a sequencecomplementary to the sequence of a first portion of a nucleic acid; andthe third container holding a second oligonucleotide having a sequencecomplementary to the sequence of a second portion of the nucleic acid.133. The kit of claim 132 further comprising a fourth container holdinga third oligonucleotide having a sequence complementary to the sequenceof a third portion of the nucleic acid, the third portion being locatedbetween the first and second portions.
 134. The kit of claim 132 furthercomprising a substrate.
 135. The kit of claim 134 further comprising: afourth container holding a binding oligonucleotide having a selectedsequence having at least two portions, the first portion beingcomplementary to at least a portion of the sequence of the secondoligonucleotide; and a fifth container holding an oligonucleotide havinga sequence complementary to the sequence of a second portion of thebinding oligonucleotide.
 136. The kit of claim 132 wherein theoligonucleotides, nanoparticles, or both bear functional groups forattachment of the oligonucleotides to the nanoparticles.
 137. The kit ofclaim 134 wherein the substrate, nanoparticles, or both bear functionalgroups for attachment of the nanoparticles to the substrate.
 138. Thekit of claim 134 wherein the substrate has nanoparticles attached to it.139. The kit of claim 132 wherein the nanoparticles are made of gold.140. A kit comprising: a substrate having oligonucleotides attachedthereto which have a sequence complementary to the sequence of a firstportion of a nucleic acid; a first container holding nanoparticleshaving oligonucleotides attached thereto, some of which have a sequencecomplementary to the sequence of a second portion of the nucleic acid;and a second container holding nanoparticles having oligonucleotidesattached thereto which have a sequence complementary to at least aportion of the sequence of the oligonucleotides attached to thenanoparticles in the first container.
 141. A kit comprising: asubstrate; a first container holding nanoparticles; a second containerholding a first oligonucleotide having a sequence complementary to thesequence of a first portion of a nucleic acid; a third container holdinga second oligonucleotide having a sequence complementary to the sequenceof a second portion of the nucleic acid; and a fourth container holdinga third oligonucleotide having a sequence complementary to at least aportion of the sequence of the second oligonucleotide.
 142. The kit ofclaim 141 wherein the oligonucleotides, nanoparticles, substrate or allbear functional groups for attachment of the oligonucleotides to thenanoparticles or for attachment of the oligonucleotides to thesubstrate.
 143. The kit of claim 141 wherein the nanoparticles are madeof gold.
 144. A kit comprising: a substrate having oligonucleotidesattached thereto which have a sequence complementary to the sequence ofa first portion of a nucleic acid; a first container holding liposomeshaving oligonucleotides attached thereto which have a sequencecomplementary to the sequence of a second portion of the nucleic acid;and a second container holding nanoparticles having at least a firsttype of oligonucleotides attached thereto, the first type ofoligonucleotides having a hydrophobic group attached to the end notattached to the nanoparticles.
 145. The kit of claim 144 wherein: thenanoparticles in the second container have a second type ofoligonucleotides attached thereto, the second type of oligonucleotideshaving a sequence complementary to the sequence of the oligonucleotideson a second type of nanoparticles; and the kit fUrther comprises: athird container holding a second type of nanoparticles havingoligonucleotides attached thereto, the oligonucleotides having asequence complementary to at least a portion of the sequence of thesecond type of oligonucleotides on the first type of nanoparticles. 146.A kit comprising at least two containers, the first container holdingparticles having oligonucleotides attached thereto which have a sequencecomplementary to the sequence of a first portion of a nucleic acid, theoligonucleotides being labeled with an energy donor on the ends notattached to the particles, the second container holding particles havingoligonucleotides attached thereto which have a sequence complementary tothe sequence of a second portion of a nucleic acid, the oligonucleotidesbeing labeled with an energy acceptor on the ends not attached to theparticles.
 147. The kit of claim 146 wherein the energy donor andacceptor are fluorescent molecules.
 148. A kit comprising at least onecontainer, the container holding a first type of particles havingoligonucleotides attached thereto which have a sequence complementary tothe sequence of a first portion of a nucleic acid, the oligonucleotidesbeing labeled with an energy donor on the ends not attached to theparticles, and a second type of particles having oligonucleotidesattached thereto which have a sequence complementary to the sequence ofa second portion of a nucleic acid, the oligonucleotides being labeledwith an energy acceptor on the ends not attached to the particles. 149.The kit of claim 148 wherein the energy donor and acceptor arefluorescent molecules.
 150. A kit comprising: a first container holdinga type of microspheres having oligonucleotides attached thereto, theoligonucleotides having a sequence complementary to a first portion ofthe sequence of a nucleic acid and being labeled with a fluorescentmolecule; and a second container holding a type of nanoparticles havingoligonucleotides attached thereto, the oligonucleotides having asequence complementary to a second portion of the sequence of thenucleic acid.
 151. The kit of claim 150 wherein the microspheres arelatex microspheres and the nanoparticles are gold nanoparticles. 152.The kit of claim 150 further comprising a microporous material.
 153. Akit comprising: a first container holding a first type of metallic orsemiconductor nanoparticles having oligonucleotides attached thereto,the oligonucleotides having a sequence complementary to a first portionof the sequence of a nucleic acid and being labeled with a fluorescentmolecule; and a second container holding a second type of metallic orsemiconductor nanoparticles having oligonucleotides attached thereto,the oligonucleotides having a sequence complementary to a second portionof the sequence of a nucleic acid and being labeled with a fluorescentmolecule.
 154. The kit of claim 153 further comprising a microporousmaterial.
 155. A kit comprising a container holding a satellite probe,the satellite probe comprising: a particle having attached theretooligonucleotides, the oligonucleotides having a first portion and asecond portion, both portions having sequences complementary to portionsof the sequence of a nucleic acid; and probe oligonucleotides hybridizedto the oligonucleotides attached to the nanoparticles, the probeoligonucleotides having a first portion and a second portion, the firstportion having a sequence complementary to the sequence of the firstportion of the oligonucleotides attached to the particles, both portionshaving sequences complementary to portions of the sequence of thenucleic acid, the probe oligonucleotides further having a reportermolecule attached to one end.
 156. A kit comprising a container holdingan aggregate probe, the aggregate probe comprising at least two types ofnanoparticles having oligonucleotides attached thereto, thenanoparticles of the aggregate probe being bound to each other as aresult of the hybridization of some of the oligonucleotides attached tothem, at least one of the types of nanoparticles of the aggregate probehaving oligonucleotides attached thereto which have a sequencecomplementary to a portion of the sequence of a nucleic acid.
 157. A kitcomprising a container holding an aggregate probe, the aggregate probecomprising at least two types of nanoparticles having oligonucleotidesattached thereto, the nanoparticles of the aggregate probe being boundto each other as a result of the hybridization of some of theoligonucleotides attached to them, at least one of the types ofnanoparticles of the aggregate probe having oligonucleotides attachedthereto which have a hydrophobic group attached to the end not attachedto the nanoparticles.
 158. An aggregate probe, the aggregate probecomprising at least two types of nanoparticles having oligonucleotidesattached thereto, the nanoparticles of the aggregate probe being boundto each other as a result of the hybridization of some of theoligonucleotides attached to them, at least one of the types ofnanoparticles of the aggregate probe having oligonucleotides attachedthereto which have a sequence complementary to a portion of the sequenceof a nucleic acid.
 159. The aggregate probe of claim 158 comprising twotypes of nanoparticles each having two types of oligonucleotidesattached thereto, the first type of oligonucleotides attached to eachtype of nanoparticles having a sequence complementary to a portion ofthe sequence of a nucleic acid, the second type of oligonucleotidesattached to the first type of nanoparticles having a sequencecomplementary to at least a portion of the sequence of the second typeof oligonucleotides attached to the second type of nanoparticles. 160.The aggregate probe of claim 158 comprising three types of nanoparticleshaving oligonucleotides attached thereto, the oligonucleotides attachedto the first type of nanoparticles having a sequence complementary to atleast a portion of the sequence of the oligonucleotides attached to thesecond type of nanoparticles, the oligonucleotides attached to thesecond type of nanoparticles having a sequence complementary to at leasta portion of the sequence of the oligonucleotides attached to the firsttype of nanoparticles, and the third type of nanoparticles having twotypes of oligonucleotides attached thereto, the first type ofoligonucleotides having a sequence complementary to a portion of thesequence of a nucleic acid, and the second type of oligonucleotideshaving a sequence complementary to at least a portion of the sequence ofthe oligonucleotides attached to the first or second type ofnanoparticles.
 161. An aggregate probe, the aggregate probe comprisingat least two types of nanoparticles having oligonucleotides attachedthereto, the nanoparticles of the aggregate probe being bound to eachother as a result of the hybridization of some of the oligonucleotidesattached to them, at least one of the types of nanoparticles of theaggregate probe having oligonucleotides attached thereto which have ahydrophobic group attached to the end not attached to the nanoparticles.162. A kit comprising a container holding a core probe, the core probecomprising at least two types of nanoparticles having oligonucleotidesattached thereto, the nanoparticles of the core probe being bound toeach other as a result of the hybridization of some of theoligonucleotides attached to them.
 163. The kit of claim 162 furthercomprising a substrate having oligonucleotides attached thereto, theoligonucleotides having a sequence complementary to a first portion ofthe sequence of a nucleic acid to be detected.
 164. The kit of claim 162or 163 further comprising a container holding a type of nanoparticleshaving two types of oligonucleotides attached thereto, the first type ofoligonucleotides having a sequence complementary to a second portion ofthe nucleic acid, and the second type of oligonucleotides havingsequence complementary to a portion of the sequence of theoligonucleotides attached to at least one of the types of nanoparticlesof the core probe.
 165. The kit of claim 162 or 163 further comprising acontainer holding a type of linking oligonucleotides comprising asequence complementary to a second portion of the sequence of thenucleic acid and a sequence complementary to a portion of the sequenceof the oligonucleotides attached to at least one of the types ofnanoparticles of the core probe.
 166. A core probe comprising at leasttwo types of nanoparticles having oligonucleotides attached thereto, thenanoparticles of the core probe being bound to each other as a result ofthe hybridization of some of the oligonucleotides attached to them. 167.A substrate having nanoparticles attached thereto.
 168. The substrate ofclaim 167 wherein the nanoparticles have oligonucleotides attachedthereto which have a sequence complementary to the sequence of a firstportion of a nucleic acid.
 169. A metallic or semiconductor nanoparticlehaving oligonucleotides attached thereto, the oligonucleotides beinglabeled with fluorescent molecules at the ends not attached to thenanoparticle.
 170. A satellite probe comprising: a particle havingattached thereto oligonucleotides, the oligonucleotides having a firstportion and a second portion, both portions having sequencescomplementary to portions of the sequence of a nucleic acid; and probeoligonucleotides hybridized to the oligonucleotides attached to thenanoparticles, the probe oligonucleotides having a first portion and asecond portion, the first portion having a sequence complementary to thesequence of the first portion of the oligonucleotides attached to theparticles, both portions having sequences complementary to portions ofthe sequence of the nucleic acid, the probe oligonucleotides furtherhaving a reporter molecule attached to one end.
 171. A method ofnanofabrication comprising providing at least one type of linkingoligonucleotide having a selected sequence, the sequence of each type oflinking oligonucleotide having at least two portions; providing one ormore types of nanoparticles having oligonucleotides attached thereto,the oligonucleotides on each of the types of nanoparticles having asequence complementary to the sequence of a portion of a linkingoligonucleotide; and contacting the linking oligonucleotides andnanoparticles under conditions effective to allow hybridization of theoligonucleotides on the nanoparticles to the linking oligonucleotides sothat a desired nanomaterial or nanostructure is formed wherein thenanoparticles are held together by oligonucleotide connectors.
 172. Themethod of claim 171 wherein at least two types of nanoparticles havingoligonucleotides attached thereto are provided, the oligonucleotides onthe first type of nanoparticles having a sequence complementary to afirst portion of the sequence of a linking oligonucleotide, and theoligonucleotides on the second te of nanoparticles having a sequencecomplementary to a second portion of the sequence of the linkingoligonucleotide.
 173. The method of claim 171 or 172 wherein thenanoparticles are metallic nanoparticles, semiconductor nanoparticles,or a combination thereof.
 174. The method of claim 173 wherein themetallic nanoparticles are made of gold, and the semiconductornanoparticles are made of CdSe/ZnS (core/shell).
 175. A method ofnanofabrication comprising: providing at least two types ofnanoparticles having oligonucleotides attached thereto, theoligonucleotides on the first type of nanoparticles having a sequencecomplementary to that of the oligonucleotides on the second of thenanoparticles; the oligonucleotides on the second type of nanoparticleshaving a sequence complementary to that of the oligonucleotides on thefirst type of nanoparticles; and contacting the first and second typesof nanoparticles under conditions effective to allow hybridization ofthe oligonucleotides on the nanoparticles to each other so that adesired nanomaterial or nanostructure is formed.
 176. The method ofclaim 175 wherein the nanoparticles are metallic nanoparticles,semiconductor nanoparticles, or a combination thereof.
 177. The methodof claim 176 wherein the metallic nanoparticles are made of gold, andthe semiconductor nanoparticles are made of CdSe/ZnS (core/shell). 178.Nanomaterials or nanostructures composed of nanoparticles havingoligonucleotides attached thereto, the nanoparticles being held togetherby oligonucleotide connectors.
 179. The nanomaterials or nanostructuresof claim 178 wherein at least some of the oligonucleotide connectors aretriple-stranded.
 180. The nanomaterials or nanostructures of claim 178wherein the nanoparticles are metallic nanoparticles, semiconductornanoparticles, or a combination thereof.
 181. The nanomaterials ornanostructures of claim 180 wherein the metallic nanoparticles are madeof gold, and the semiconductor nanoparticles are made of CdSe/ZnS(core/shell).
 182. A composition comprising at least two types ofnanoparticles having oligonucleotides attached thereto, theoligonucleotides on the first type of nanoparticles having a sequencecomplementary to the sequence of a first portion of a nucleic acid or alinking oligonucleotide, the oligonucleotides on the second type ofnanoparticles having a sequence complementary to the sequence of asecond portion of the nucleic acid or linking oligonucleotide.
 183. Thecomposition of claim 182 wherein the nanoparticles are metallicnanoparticles, semiconductor nanoparticles, or a combination thereof.184. The composition of claim 183 wherein the metallic nanoparticles aremade of gold, and the semiconductor nanoparticles are made of CdSe/ZnS(core/shell).
 185. An assembly of containers comprising: a firstcontainer holding nanoparticles having oligonucleotides attachedthereto, and a second container holding nanoparticles havingoligonucleotides attached thereto, the oligonucleotides attached to thenanoparticles in the first container having a sequence complementary tothat of the oligonucleotides attached to the nanoparticles in the secondcontainer, the oligonucleotides attached to the nanoparticles in thesecond container having a sequence complementary to that of theoligonucleotides attached to the nanoparticles in the second container.186. The assembly of claim 185 wherein the nanoparticles are metallicnanoparticles, semiconductor nanoparticles, or a combination thereof.187. The assembly of claim 186 wherein the metallic nanoparticles aremade of gold, and the semiconductor nanoparticles are made of CdSe/ZnS(core/shell).
 188. A nanoparticle having a plurality of differentoligonucleotides attached thereto.
 189. A method of separating aselected nucleic acid having at least two portions from other nucleicacids, the method comprising: providing two or more types ofnanoparticles having oligonucleotides attached thereto, theoligonucleotides on each of the types of nanoparticles having a sequencecomplementary to the sequence of one of the portions of the selectednucleic acid; and contacting the nucleic acids and nanoparticles underconditions effective to allow hybridization of the oligonucleotides onthe nanoparticles with the selected nucleic acid so that thenanoparticles hybridized to the selected nucleic acid aggregate andprecipitate.
 190. A method of binding oligonucleotides to chargednanoparticles to produce stable nanoparticle-oligonucleotide conjugates,the method comprising: providing oligonucleotides having covalentlybound thereto a moiety comprising a functional group which can bind tothe nanoparticles; contacting the oligonucleotides and the nanoparticlesin water for a period of time sufficient to allow at least some of theoligonucleotides to bind to the nanoparticles; adding at least one saltto the water to form a salt solution, the ionic strength of the saltsolution being sufficient to overcome at least partially theelectrostatic attraction or repulsion of the oligonucleotides for thenanoparticles and the electrostatic repulsion of the oligonucleotidesfor each other; and contacting the oligonucleotides and nanoparticles inthe salt solution for an additional period of time sufficient to allowsufficient additional oligonucleotides to bind to the nanoparticles toproduce the stable nanoparticle-oligonucleotide conjugates.
 191. Themethod of claim 190 wherein the nanoparticles are metal nanoparticles orsemiconductor nanoparticles.
 192. The method of claim 191 wherein thenanoparticles are gold nanoparticles.
 193. The method of claim 192wherein the moiety comprising a functional group which can bind to thenanoparticles is an alkanethiol.
 194. The method of claim 190 whereinall of the salt is added to the water in a single addition.
 195. Themethod of claim 190 wherein the salt is added gradually over time. 196.The method of claim 190 wherein the salt is selected from the groupconsisting of sodium chloride, magnesium chloride, potassium chloride,ammonium, chloride, sodium, acetate, ammonium acetate, a combination oftwo or more of these salts, one of these salts in a phosphate buffer,and a combination of two or more these salts in a phosphate buffer. 197.The method of claim 196 wherein the salt is sodium chloride in aphosphate buffer.
 198. The method of claim 190 whereinnanoparticle-oligonucleotide conjugates are produced which have theoligonucleotides present on surface of the nanoparticles at a surfacedensity of at least 10 picomoles/cm².
 199. The method of claim 198wherein the oligonucleotides are present on surface of the nanoparticlesat a surface density of at least 15 picomoles/cm².
 200. The method ofclaim 199 wherein the oligonucleotides are present on surface of thenanoparticles at a surface density of from about 15 picomoles/cm² toabout 40 picomoles/cm².
 201. A method of binding oligonucleotides tonanoparticles to produce nanoparticle-oligonucleotide conjugates, themethod comprising: providing oligonucleotides, the oligonucleotidescomprising at least one type of recognition oligonucleotides, each ofthe recognition oligonucleotides comprising a spacer portion and arecognition portion, the spacer portion being designed so that it canbind to the nanoparticles; and contacting the oligonucleotides and thenanoparticles under conditions effective to allow at least some of therecognition oligonucleotides to bind to the nanoparticles to produce thenanoparticle-oligonucleotide conjugates.
 202. The method of claim 201wherein each of the spacer portions of the recognition oligonucleotideshas a moiety covalently bound thereto, the moiety comprising afunctional group which can bind to the nanoparticles
 203. The method ofclaim 201 wherein the nanoparticles are metal nanoparticles orsemiconductor nanoparticles.
 204. The method of claim 203 wherein thenanoparticles are gold nanoparticles.
 205. The method of claim 204wherein the spacer portion comprises at least about nucleotides. 206.The method of claim 205 wherein the spacer portion comprises from about10 to about 30 nucleotides.
 207. The method of claim 206 wherein thebases of the nucleotides of the spacer are all adenines, all thymines,all cytosines, all uracils, or all guanines.
 208. A method of bindingoligonucleotides to nanoparticles to producenanoparticle-oligonucleotide conjugates, the method comprising:providing oligonucleotides, the oligonucleotides comprising: a type ofrecognition oligonucleotides; and a type of diluent oligonucleotides;contacting the oligonucleotides with the nanoparticles under conditionseffective to allow at least some of each of the types ofoligonucleotides to bind to the nanoparticles to produce thenanoparticle-oligonucleotide conjugates.
 209. The method of claim 208wherein the nanoparticles are metal nanoparticles or semiconductornanoparticles.
 210. The method of claim 209 wherein the nanoparticlesare gold nanoparticles.
 211. The method of claim 208 wherein each of therecognition oligonucleotides comprises a spacer portion and arecognition portion, the spacer portion being designed so that it canbind to the nanoparticles.
 212. The method of claim 211 wherein each ofthe spacer portions of the recognition oligonucleotides has a moietycovalently bound thereto, the moiety comprising a functional group whichcan bind to the nanoparticles.
 213. The method of claim 211 wherein thespacer portions of the recognition oligonucleotides comprises at leastabout 10 nucleotides.
 214. The method of claim 213 wherein the spacerportions of the recognition oligonucleotides comprises from about 10nucleotides to about 30 nucleotides.
 215. The method of claim 211wherein the bases of the nucleotides of the spacer are all adenines, allthymines, all cytosines, all uracils or all guanines.
 216. The method ofclaim 211 wherein the diluent oligonucleotides contain about the samenumber of nucleotides as are contained in the spacer portions of therecognition oligonucleotides.
 217. The method of claim 216 wherein thesequence of the diluent oligonucleotides is the same as the sequence ofthe spacer portions of the recognition oligonucleotides.
 218. The methodof claim 208 wherein the oligonucleotides comprise at least two types ofrecognition oligonucleotides.
 219. A method of binding oligonucleotidesto charged nanoparticles to produce nanoparticle-oligonucleotideconjugates, the method comprising: providing oligonucleotides havingcovalently bound thereto a moiety comprising a functional group whichcan bind to the nanoparticles, the oligonucleotides comprising: a typeof recognition oligonucleotides; and a type of diluent oligonucleotides;contacting the oligonucleotides with the nanoparticles in water for aperiod of time sufficient to allow at least some of each of the types ofoligonucleotides to bind to the nanoparticles; adding at least one saltto the water to form a salt solution, the ionic strength of the saltsolution being sufficient to overcome at least partially theelectrostatic attraction or repulsion of the oligonucleotides for thenanoparticles and the electrostatic repulsion of the oligonucleotidesfor each other; and contacting the oligonucleotides and nanoparticles inthe salt solution for an additional period of time sufficient to allowadditional oligonucleotides of each of the types of oligonucleotides tobind to the nanoparticles to produce the nanoparticle-oligonucleotideconjugates.
 220. The method of claim 219 wherein the nanoparticles aremetal nanoparticles or semiconductor nanoparticles.
 221. The method ofclaim 220 wherein the nanoparticles are gold nanoparticles.
 222. Themethod of claim 221 wherein the moiety comprising a functional groupwhich can bind to the nanoparticles is an alkanethiol.
 223. The methodof claim 219 wherein all of the salt is added to the water in a singleaddition.
 224. The method of claim 219 wherein the salt is addedgradually over time.
 225. The method of claim 219 wherein the salt isselected from the group consisting of sodium chloride, magnesiumchloride, potassium chloride, ammonium, chloride, sodium, acetate,ammonium acetate, a combination of two or more of these salts, one ofthese salts in a phosphate buffer, and a combination of two or morethese salts in a phosphate buffer.
 226. The method of claim 225 whereinthe salt is sodium chloride in a phosphate buffer.
 227. The method ofclaim 219 wherein nanoparticle-oligonucleotide conjugates are producedwhich have the oligonucleotides are present on surface of thenanoparticles at a surface density of at least 10 picomoles/cm². 228.The method ofclaim 227 wherein the oligonucleotides are present onsurface of the nanoparticles at a surface density of at least 15picomoles/cm².
 229. The method of claim 228 wherein the oligonucleotidesare present on surface of the nanoparticles at a surface density of fromabout 15 picomoles/cm² to about 40 picomoles/cm².
 230. The method ofclaim 219 wherein each of the recognition oligonucleotides comprises aspacer portion and a recognition portion, the spacer portion havingattached to it the moiety comprising a functional group which can bindto the nanoparticles.
 231. The method of claim 230 wherein the spacerportion comprises at least about 10 nucleotides.
 232. The method ofclaim 231 wherein the spacer portion comprises from about 10 to about 30nucleotides.
 233. The method of claim 230 wherein the bases of thenucleotides of the spacers are all adenines, all thymines, allcytosines, all uracils, or all guanines.
 234. The method of claim 230wherein the diluent oligonucleotides contain about the same number ofnucleotides as are contained in the spacer portions of the recognitionoligonucleotides.
 235. The method of claim 234 wherein the sequence ofthe diluent oligonucleotides is the same as the sequence of the spacerportions of the recognition oligonucleotides.
 236. The method of claim219 wherein the oligonucleotides comprise at least two types ofrecognition oligonucleotides.
 237. Nanoparticle-oligonucleotideconjugates which are nanoparticles having oligonucleotides attached tothem, the oligonucleotides being present on surface of the nanoparticlesat a surface density sufficient so that the conjugates are stable, atleast some of the oligonucleotides having a sequence complementary to atleast one portion of the sequence of a nucleic acid or anotheroligonucleotide.
 238. The conjugates of claim 237 wherein theoligonucleotides are present on surface of the nanoparticles at asurface density of at least 10 picomoles/cm²
 239. The nanoparticles ofclaim 238 wherein the oligonucleotides are present on surface of thenanoparticles at a surface density of at least 15 picomoles/cm². 240.The nanoparticles of claim 239 wherein the oligonucleotides are presenton surface of the nanoparticles at a surface density of from about 15picomoles/cm² to about 40 picomoles/cm².
 241. The nanoparticles of claim237 wherein the nanoparticles are metal nanoparticles or semiconductornanoparticles.
 242. The nanoparticles of claim 241 wherein thenanoparticles are gold nanoparticles.
 243. Nanoparticles havingoligonucleotides attached to them, the oligonucleotides comprising atleast one type of recognition oligonucleotides, each of the recognitionoligonucleotides comprising a spacer portion and a recognition portion,the spacer portion being designed so that it is bound to thenanoparticles, the recognition portion having a sequence complementaryto at least one portion of the sequence of a nucleic acid or anotheroligonucleotide.
 244. The nanoparticles of claim 243 wherein the spacerportion has a moiety covalently bound to it, the moiety comprising afunctional group through which the spacer portion is bound to thenanoparticles.
 245. The nanoparticles of claim 243 wherein the spacerportion comprises at least about 10 nucleotides.
 246. The nanoparticlesof claim 245 wherein the spacer portion comprises from about 10 to about30 nucleotides.
 247. The nanoparticles of claim 243 wherein the bases ofthe nucleotides of the spacer portion are all adenines, all thymines,all cytosines, all uracils or all guanines.
 248. The nanoparticles ofclaim 243 wherein the oligonucleotides are present on surface of thenanoparticles at a surface density of at least 10 picomoles/cm². 249.The nanoparticles of claim 248 wherein the oligonucleotides are presenton surface of the nanoparticles at a surface density of at least 15picomoles/cm².
 250. The nanoparticles of claim 249 wherein theoligonucleotides are present on surface of the nanoparticles at asurface density of from about 15 picomoles/cm² to about 40picomoles/cm².
 251. The nanoparticles of claim 243 wherein thenanoparticles are metal nanoparticles or semiconductor nanoparticles.252. The method of claim 251 wherein the nanoparticles are goldnanoparticles.
 253. Nanoparticles having oligonucleotides attached tothem, the oligonucleotides comprising: at least one type of recognitionoligonucleotides, each of the types of recognition oligonucleotidescomprising a sequence complementary to at least one portion of thesequence of a nucleic acid or another oligonucleotide; and a type ofdiluent oligonucleotides.
 254. The nanoparticles of claim 253 wherein,each of the recognition oligonucleotides comprises a spacer portion anda recognition portion, the spacer portion being designed so that it isbound to the nanoparticles, the recognition portion having a sequencecomplementary to at least one portion of the sequence of a nucleic acidor another oligonucleotide.
 255. The nanoparticles of claim 254 whereinthe spacer portion has a moiety covalently bound to it, the moietycomprising a functional group through which the spacer portion is boundto the nanoparticles.
 256. The nanoparticles of claim 254 wherein thespacer portion comprises at least about 10 nucleotides.
 257. Thenanoparticles of claim 256 wherein the spacer portion comprises fromabout 10 to about 30 nucleotides.
 258. The nanoparticles of claim 254wherein the bases of the nucleotides of the spacer portion are alladenines, all thymines, all cytosines, all uracils or all guanines. 259.The nanoparticles of claim 253 wherein the oligonucleotides are presenton surface of the nanoparticles at a surface density of at least 10picomoles/cm².
 260. The nanoparticles of claim 259 wherein theoligonucleotides are present on surface of the nanopaiticles at asurface density of at least 15 picomoles/cm².
 261. The nanoparticles ofclaim 260 wherein the oligonucleotides are present on surface of thenanoparticles at a surface density of from about 15 picomoles/cm² toabout 40 picomoles/cm².
 262. The nanoparticles of claim 254 wherein thediluent oligonucleotides contain about the same number of nucleotides asare contained in the spacer portions of the recognitionoligonucleotides.
 263. The nanoparticles of claim 262 wherein thesequence of the diluent oligonucleotides is the same as that of thespacer portions of the recognition oligonucleotides.
 264. Thenanoparticles of claim 253 wherein the nanoparticles are metalnanoparticles or semiconductor nanoparticles.
 265. The nanoparticles ofclaim 264 wherein the nanoparticles are gold nanoparticies.
 266. Amethod of detecting a nucleic acid comprising: contacting the nucleicacid with at least one type of nanoparticle-oligonucleotide conjugatesaccording to any one of claims 237-242 under conditions effective toallow hybridization of the oligonucleotides on the nanoparticles withthe nucleic acid; and observing a detectable change brought about byhybridization of the oligonucleotides on the nanoparticles with thenucleic acid.
 267. A method of detecting a nucleic acid comprising:contacting the nucleic acid with at least one type of nanoparticlesaccording to any one of claims 243-265 under conditions effective toallow hybridization of at least one of the types of recognitionoligonucleotides on the nanoparticles with the nucleic acid; andobserving a detectable change brought about by hybridization of therecognition oligonucleotides with the nucleic acid.
 268. A method ofdetecting a nucleic acid having at least two portions comprising:providing a type of nanoparticle-oligonucleotide conjugates according toany one of claims 237-242, the oligonucleotides on each nanoparticlehaving a sequence complementary to the sequence of at least two portionsof the nucleic acid; contacting the nucleic acid and the conjugatesunder conditions effective to allow hybridization of theoligonucleotides on the nanoparticles with the two or more portions ofthe nucleic acid; and observing a detectable change brought about byhybridization of the oligonucleotides on the nanoparticles with thenucleic acid.
 269. A method of detecting a nucleic acid having at leasttwo portions comprising: contacting the nucleic acid with at least twotypes of nanoparticle-oligonucleotide conjugates according to any one ofclaims 237-240, the oligonucleotides on the nanoparticles of the firsttype of conjugates having a sequence complementary to a first portion ofthe sequence of the nucleic acid, the oligonucleotides on thenanoparticles of the second type of conjugates having a sequencecomplementary to a second portion of the sequence of the nucleic acid,the contacting taking place under conditions effective to allowhybridization of the oligonucleotides on the nanoparticles with thenucleic acid; and observing a detectable change brought about byhybridization of the oligonucleotides on the nanoparticles with thenucleic acid.
 270. The method of claim 269 wherein the contactingconditions include freezing and thawing.
 271. The method of claim 269wherein the contacting conditions include heating.
 272. The method ofclaim 269 wherein the detectable change is observed on a solid surface.273. The method of claim 269 wherein the detectable change is a colorchange observable with the naked eye.
 274. The method of claim 273wherein the color change is observed on a solid surface.
 275. The methodof claim 269 wherein the nanoparticles are metal nanoparticles orsemiconductor nanoparticles.
 276. The method of claim 269 wherein thenanoparticles are gold nanoparticles.
 277. The method of claim 269wherein the oligonucleotides attached to the nanoparticles are labeledon their ends not attached to the nanoparticles with molecules thatproduce a detectable change upon hybridization of the oligonucleotideson the nanoparticles with the nucleic acid.
 278. The method of claim 277wherein the nanoparticles are metallic or semiconductor nanoparticlesand the oligonucleotides attached to the nanoparticles are labeled withfluorescent molecules.
 279. The method of claim 269 wherein: the nucleicacid has a third portion located between the first and second portions,and the sequences of the oligonucleotides on the nanoparticles do notinclude sequences complementary to this third portion of the nucleicacid; and the nucleic acid is further contacted with a filleroligonucleotide having a sequence complementary to this third portion ofthe nucleic acid, the contacting taking place under conditions effectiveto allow hybridization of the filler oligonucleotide with the nucleicacid.
 280. The method of claim 269 wherein the nucleic acid is viral RNAor DNA.
 281. The method of claim 269 wherein the nucleic acid isa geneassociated with a disease.
 282. The method of claim 269 wherein thenucleic acid is a bacterial DNA.
 283. The method of claim 269 whereinthe nucleic acid is a fungal DNA.
 284. The method of claim 269 whereinthe nucleic acid is a synthetic DNA, a synthetic RNA, astructurally-modified natural or synthetic RNA, or astructurally-modified natural or synthetic DNA.
 285. The method of claim269 wherein the nucleic acid is from a biological source.
 286. Themethod of claim 269 wherein the nucleic acid is a product of apolymerase chain reaction amplification.
 287. The method of claim 269wherein the nucleic acid is contacted with the first and second types ofconjugates simultaneously.
 288. The method of claim 269 wherein thenucleic acid is contacted and hybridized with the oligonucleotides onthe nanoparticles of first type of conjugates before being contactedwith the second type of conjugates.
 289. The method of claim 288 whereinthe first type of conjugates is attached to a substrate.
 290. The methodof claim 269 wherein the nucleic acid is double-stranded andhybridization with the oligonucleotides on the nanoparticles results inthe production of a triple-stranded complex.
 291. A method of detectinga nucleic acid having at least two portions comprising: providing a typeof nanoparticles according to any one of claims 243-252 havingrecognition oligonucleotides attached thereto, the recognitionoligonucleotides on each nanoparticle comprising a sequencecomplementary to the sequence of at least two portions of the nucleicacid; contacting the nucleic acid and the nanoparticles under conditionseffective to allow hybridization of the oligonucleotides on thenanoparticles with the two or more portions of the nucleic acid; andobserving a detectable change brought about by hybridization of theoligonucleotides on the nanoparticles with the nucleic acid.
 292. Amethod of detecting nucleic acid having at least two portionscomprising: contacting the nucleic acid with at least two types ofnanoparticles according to any one of claims 243-250 having recognitionoligonucleotides attached thereto, the recognition oligonucleotides onthe first type of nanoparticles comprising a sequence complementary to afirst portion of the sequence of the nucleic acid, the recognitionoligonucleotides on the second type of nanoparticles comprising asequence complementary to a second portion of the sequence of thenucleic acid, the contacting taking place under conditions effective toallow hybridization of the recognition oligonucleotides on thenanoparticles with the nucleic acid; and observing a detectable changebrought about by hybridization of the recognition oligonucleotides onthe nanoparticles with the nucleic acid.
 293. The method of claim 292wherein the contacting conditions include freezing and thawing.
 294. Themethod of claim 292 wherein the contacting conditions include heating.295. The method of claim 292 wherein the detectable change is observedon a solid surface.
 296. The method of claim 292 wherein the detectablechange is a color change observable with the naked eye.
 297. The methodof claim 296 wherein the color change is observed on a solid surface.298. The method of claim 292 wherein the nanoparticles are metalnanoparticles or semiconductor nanoparticles.
 299. The method of claim298 wherein the nanoparticles are made of gold.
 300. The method of claim292 wherein the recognition oligonucleotides attached to thenanoparticles are labeled on their ends not attached to thenanoparticles with molecules that produce a detectable change uponhybridization of the oligonucleotides on the nanoparticles with thenucleic acid.
 301. The method of claim 300 wherein the nanoparticles aremetallic or semiconductor nanoparticles and the oligonucleotidesattached to the nanoparticles are labeled with fluorescent molecules.302. The method of claim 292 wherein: the nucleic acid has a thirdportion located between the first and second portions, and the sequencesof the oligonucleotides on the nanoparticles do not include sequencescomplementary to this third portion of the nucleic acid; and the nucleicacid is further contacted with a filler oligonucleotide having asequence complementary to this third portion of the nucleic acid, thecontacting taking place under conditions effective to allowhybridization of the filler oligonucleotide with the nucleic acid. 303.The method of claim 292 wherein the nucleic acid is viral RNA or DNA.304. The method of claim 292 wherein the nucleic acid is a geneassociated with a disease.
 305. The method of claim 292 wherein thenucleic acid is a bacterial DNA.
 306. The method of claim 292 whereinthe nucleic acid is a fingal DNA.
 307. The method of claim 292 whereinthe nucleic acid is a synthetic DNA, a synthetic RNA, astructurally-modified natural or synthetic RNA, or astructurally-modified natural or synthetic DNA.
 308. The method of claim292 wherein the nucleic acid is from a biological source.
 309. Themethod of claim 292 wherein the nucleic acid is a product of apolymerase chain reaction amplification.
 310. The method of claim 292wherein the nucleic acid is contacted with the first and second types ofnanoparticles simultaneously.
 311. The method of claim 292 wherein thenucleic acid is contacted and hybridized with the oligonucleotides onthe first type of nanoparticles before being contacted with the secondtype of nanoparticles.
 312. The method of claim 31 1 wherein the firsttype of nanoparticles is attached to a substrate.
 313. The method ofclaim 292 wherein the nucleic acid is double-stranded and hybridizationwith the oligonucleotides on the nanoparticles results in the productionof a triple-stranded complex.
 314. A method of detecting a nucleic acidhaving at least two portions comprising: providing a type ofnanoparticles according to any one of claims 253-265 having recognitionoligonucleotides attached thereto, the recognition oligonucleotides oneach nanoparticle comprising a sequence complementary to the sequence ofat least two portions of the nucleic acid; contacting the nucleic acidand the nanoparticles under conditions effective to allow Hybridizationof the recognition oligonucleotides on the nanoparticles with the two ormore portions of the nucleic acid; and observing a detectable changebrought about by hybridization of the recognition oligonucleotides onthe nanoparticles with the nucleic acid.
 315. A method of detectingnucleic acid having at least two portions comprising: contacting thenucleic acid with at least two types of nanoparticles according to anyone of claims 253-263 having recognition oligonucleotides attachedthereto, the recognition oligonucleotides on the first type ofnanoparticles comprising a sequence complementary to a first portion ofthe sequence of the nucleic acid, the recognition oligonucleotides onthe second type of nanoparticles comprising a sequence complementary toa second portion of the sequence of the nucleic acid, the contactingtaking place under conditions effective to allow hybridization of therecognition oligonucleotides on the nanoparticles with the nucleic acid;and observing a detectable change brought about by hybridization of therecognition oligonucleotides on the nanoparticles with the nucleic acid.316. The method of claim 315 wherein the contacting conditions includefreezing and thawing.
 317. The method of claim 315 wherein thecontacting conditions include heating.
 318. The method of claim 315wherein the detectable change is observed on a solid surface.
 319. Themethod of claim 315 wherein the detectable change is a color changeobservable with the naked eye.
 320. The method of claim 319 wherein thecolor change is observed on a solid surface.
 321. The method of claim315 wherein the nanoparticles are metal nanoparticles or semiconductornanoparticles.
 322. The method of claim 321 wherein the nanoparticlesare made of gold.
 323. The method of claim 315 wherein the recognitionoligonucleotides attached to the nanoparticles are labeled on their endsnot attached to the nanoparticles with molecules that produce adetectable change upon hybridization of the recognition oligonucleotideson the nanoparticles with the nucleic acid.
 324. The method of claim 323wherein the nanoparticles are metallic or semiconductor nanoparticlesand the recognition oligonucleotides attached to the nanoparticles arelabeled with fluorescent molecules.
 325. The method of claim 315wherein: the nucleic acid has a third portion located between the firstand second portions, and the sequences of the oligonucleotides on thenanoparticles do not include sequences complementary to this thirdportion of the nucleic acid; and the nucleic acid is further contactedwith a filler oligonucleotide having a sequence complementary to thisthird portion of the nucleic acid, the contacting taking place underconditions effective to allow hybridization of the filleroligonucleotide with the nucleic acid.
 326. The method of claim 315wherein the nucleic acid is viral RNA or DNA.
 327. The method of claim315 wherein the nucleic acid is a gene associated with a disease. 328.The method of claim 315 wherein the nucleic acid is a bacterial DNA.329. The method of claim 315 wherein the nucleic acid is a fungal DNA.330. The method of claim 315 wherein the nucleic acid is a syntheticDNA, a synthetic RNA, a structurally-modified natural or synthetic RNA,or a structurally-modified natural or synthetic DNA.
 331. The method ofclaim 315 wherein the nucleic acid is from a biological source.
 332. Themethod of claim 315 wherein the nucleic acid is a product of apolymerase chain reaction amplification.
 333. The method of claim 315wherein the nucleic acid is contacted with the first and second types ofnanoparticles simultaneously.
 334. The method of claim 315 wherein thenucleic acid is contacted and hybridized with the recognitionoligonucleotides on the first type of nanoparticles before beingcontacted with the second type of nanoparticles.
 335. The method ofclaim 334 wherein the first type of nanoparticles is attached to asubstrate.
 336. The method of claim 315 wherein the nucleic acid isdouble-stranded and hybridization with the oligonucleotides on thenanoparticles results in the production of a triple-stranded complex.337. A method of detecting a nucleic acid having at least two portionscomprising: (a) contacting the nucleic acid with a substrate havingoligonucleotides attached thereto, the oligonucleotides having asequence complementary to a first portion of the sequence of saidnucleic acid, the contacting taking place under conditions effective toallow hybridization of the oligonucleotides on the substrate with saidnucleic acid; (b) contacting said nucleic acid bound to the substratewith a first type of nanoparticle-oligonucleotide conjugates accordingto any one of claims 237-240, at least one of the types ofoligonucleotides attached to the nanoparticles of the conjugates havinga sequence complementary to a second portion of the sequence of saidnucleic acid, the contacting taking place under conditions effective toallow hybridization of the oligonucleotides attached to thenanoparticles of the conjugates with said nucleic acid; and (c)observing a detectable change.
 338. The method of claim 337 furthercomprising: (d) contacting the first type ofnanoparticle-oligonucleotide conjugates bound to the substrate with asecond type of nanoparticle-oligonucleotide conjugates according to anyone of claims 237-240, at least one of the types of oligonucleotidesattached to the nanoparticles of the second type of conjugates having asequence complementary to the sequence of one of the types ofoligonucleotides attached to the nanoparticles of the first type ofconjugates, the contacting taking place under conditions effective toallow hybridization of the oligonucleotides attached to thenanoparticles of the first and second types of conjugates; and (e)observing the detectable change.
 339. The method of claim 338 wherein atleast one of the types of oligonucleotides on the nanoparticles of thefirst type of conjugates has a sequence complementary to the sequence ofat least one of the types of oligonucleotides on the nanoparticles ofthe second type of conjugates and the method further comprises: (f)contacting the second type of conjugates bound to the substrate with thefirst type of conjugates, the contacting taking place under conditionseffective to allow hybridization of the oligonucleotides on thenanoparticles of the first and second types of conjugates; and (g)observing the detectable change.
 340. The method of claim 339 whereinstep (d) or steps (d) and (f) are repeated one or more times and thedetectable change is observed.
 341. The method of claim 337 furthercomprising: (d) providing a type of binding oligonucleotides having asequence comprising at least two portions, the first portion beingcomplementary to at least one of the types of oligonucleotides attachedto the nanoparticles of the first type of conjugates; (e) contacting thebinding oligonucleotides with the first type of conjugates bound to thesubstrate, the contacting taking place under conditions effective toallow hybridization of the binding oligonucleotides with theoligonucleotides on the nanoparticles of the first type of conjugates;(f) providing a second type of nanoparticle-oligonucleotide conjugatesaccording to any one of claims 237-240, at least one of the types ofoligonucleotides attached to the nanoparticles of the second type ofconjugates having a sequence complementary to the second portion of thesequence of the binding oligonucleotides; (g) contacting the bindingoligonucleotides bound to the substrate with the second type ofconjugates, the contacting taking place under conditions effective toallow hybridization of the oligonucleotides attached to thenanoparticles of the second type of conjugates with the bindingoligonucleotides; and (h) observing the detectable change.
 342. Themethod of claim 341 further comprising: (i) contacting the second typeof conjugates bound to the substrate with the binding oligonucleotides,the contacting taking place under conditions effective to allowhybridization of the binding oligonucleotides with the oligonucleotideson the nanoparticles of the second type of conjugates; (j) contactingthe binding oligonucleotides bound to the substrate with the first typeof conjugates, the contacting taking place under conditions effective toallow hybridization of the oligonucleotides on the nanoparticles of thefirst type of conjugates with the binding oligonucleotides; and (k)observing the detectable change.
 343. The method of claim 342 whereinsteps (e) and (g) or steps (e), (g), (i) and (j) are repeated one ormore times, and the detectable change is observed.
 344. The method ofclaim 337 wherein the substrate is a transparent substrate or an opaquewhite substrate.
 345. The method of claim 344 wherein the detectablechange is the formation of dark areas on the substrate.
 346. The methodof claim 337 wherein the nanoparticles of the conjugates are metalnanoparticles or semiconductor nanoparticles.
 347. The method of claim346 wherein the nanoparticles of the conjugates are made of gold orsilver.
 348. The method of claim 337 wherein the substrate has aplurality of types of oligonucleotides attached to it in an array toallow for the detection of multiple portions of a single nucleic acid,the detection of multiple different nucleic acids, or both.
 349. Themethod of claim 337 wherein the substrate is contacted with silver stainto produce the detectable change.
 350. The method of claim 348 whereinthe substrate is contacted with silver stain to produce the detectablechange.
 351. The method of claim 337 wherein the detectable change isobserved with an optical scanner
 352. The method of claim 351 whereinthe device is a flatbed scanner.
 353. The method of claim 351 whereinthe scanner is linked to a computer loaded with software capable ofcalculating greyscale measurements, and the greyscale measurements arecalculated. to provide a quantitative measure of the amount of nucleicacid detected.
 354. The method of claim 337 wherein the oligonucleotidesattached to the substrate are located between two electrodes, thenanoparticles of the conjugates are made of a material which is aconductor of electricity, and the detectable change is a change inconductivity.
 355. The method of claim 354 wherein the electrodes aremade of gold, and the nanoparticles are made of gold.
 356. The method ofclaim 354 wherein the substrate is contacted with silver stain toproduce the change in conductivity.
 357. The method of claim 348 whereineach of the plurality of oligonucleotides attached to the substrate inthe array is located between two electrodes, the nanoparticles are madeof a material which is a conductor of electricity, and the detectablechange is a change in conductivity.
 358. The method of claim 357 whereinthe electrodes are made of gold, and the nanoparticles are made of gold.359. The method of claim 357 wherein the substrate is contacted withsilver stain to produce the change in conductivity.
 360. A method ofdetecting a nucleic acid having at least two portions comprising: (a)contacting the nucleic acid with a substrate having oligonucleotidesattached thereto, the oligonucleotides having a sequence complementaryto a first portion of the sequence of said nucleic acid, the contactingtaking place under conditions effective to allow hybridization of theoligonucleotides on the substrate with said nucleic acid; (b) contactingsaid nucleic acid bound to the substrate with a first type ofnanoparticles according to any one of claims 243-250 having one or moretypes of recognition oligonucleotides attached thereto, at least one ofthe types of recognition oligonucleotides comprising a sequencecomplementary to a second portion of the sequence of said nucleic acid,the contacting taking place under conditions effective to allowhybridization of the oligonucleotides on the nanoparticles with saidnucleic acid; and (c) observing a detectable change.
 361. The method ofclaim 360 further comprising: (d) contacting the first type ofnanoparticles bound to the substrate with a second type of nanoparticlesaccording to any one of claims 243-250 having recognitionoligonucleotides attached thereto, at least one of the types ofrecognition oligonucleotides on the second type of nanoparticlescomprising a sequence complementary to the sequence of one of the typesof oligonucleotides on the first type of nanoparticles, the contactingtaking place under conditions effective to allow hybridization of theoligonucleotides on the first and second types of nanoparticles; and (e)observing the detectable change.
 362. The method of claim 360 wherein atleast one of the types of recognition oligonucleotides on the first typeof nanoparticles has a sequence complementary to the sequence of atleast one of the types of oligonucleotides on the second type ofnanoparticles and the method further comprises: (f) contacting thesecond type of nanoparticles bound to the substrate with the first typeof nanoparticles, the contacting taking place under conditions effectiveto allow hybridization of the oligonucleotides on the first and secondtypes of nanoparticles; and (g) observing the detectable change. 363.The method of claim 362 wherein step (d) or steps (d) and (f) arerepeated one or more times and the detectable change is observed. 364.The method of claim 360 further comprising: (d) providing a type ofbinding oligonucleotides having a sequence comprising at least twoportions, the first portion being complementary to at least one of thetypes of oligonucleotides on the first type of nanoparticles; (e)contacting the binding oligonucleotides with the first type ofnanoparticles bound to the substrate, the contacting taking place underconditions effective to allow hybridization of the bindingoligonucleotides with the oligonucleotides on the first type ofnanoparticles; (f) providing a second type of nanoparticles according toany one of claims 243-250 having recognition oligonucleotides attachedthereto, at least one of the types of recognition oligonucleotides onthe second type of nanoparticles comprising a sequence complementary tothe second portion of the sequence of the binding oligonucleotides; (g)contacting the binding oligonucleotides bound to the substrate with thesecond type of nanoparticles, the contacting taking place underconditions effective to allow hybridization of the oligonucleotides onthe second type of nanoparticles with the binding oligonucleotides; and(h) observing the detectable change.
 365. The method of claim 364further comprising: (i) contacting the second type of nanoparticlesbound to the substrate with the binding oligonucleotides, the contactingtaking place under conditions effective to allow hybridization of thebinding oligonucleotides with the oligonucleotides on the second type ofnanoparticles; (j) contacting the binding oligonucleotides bound to thesubstrate with the first type of nanoparticles, the contacting takingplace under conditions effective to allow hybridization of theoligonucleotides on the first type of nanoparticles with the bindingoligonucleotides; and (k) observing the detectable change.
 366. Themethod of claim 365 wherein steps (e) and (g) or steps (e), (g), (i) and(j) are repeated one or more times, and the detectable change isobserved.
 367. The method of claim 360 wherein the substrate is atransparent substrate or an opaque white substrate.
 368. The method ofclaim 3 67 wherein the detectable change is the formation of dark areason the substrate.
 369. The method of claim 360 wherein the nanoparticlesare metal nanoparticles or semiconductor nanoparticles.
 370. The methodof claim 369 wherein the nanoparticles are made of gold or silver. 371.The method of claim 360 wherein the substrate has a plurality of typesof oligonucleotides attached to it in an array to allow for thedetection of multiple portions of a single nucleic acid, the detectionof multiple different nucleic acids, or both.
 372. The method of claim360 wherein the substrate is contacted with silver stain to produce thedetectable change.
 373. The method of claim 371 wherein the substrate iscontacted with silver stain to produce the detectable change.
 375. Themethod of claim 360 wherein the detectable change is observed with anoptical scanner
 376. The method of claim 375 wherein the device is aflatbed scanner.
 377. The method of claim 375 wherein the scanner islinked to a computer loaded with software capable of calculatinggreyscale measurements, and the greyscale measurements are calculated.to provide a quantitative measure of the amount of nucleic aciddetected.
 378. The method of claim 360 wherein the oligonucleotidesattached to the substrate are located between two electrodes, thenanoparticles are made of a material which is a conductor ofelectricity, and the detectable change is a change in conductivity. 379.The method of claim 378 wherein the electrodes are made of gold, and thenanoparticles are made of gold.
 380. The method of claim 378 wherein thesubstrate is contacted with silver stain to produce the change inconductivity.
 381. The method of claim 371 wherein each of the pluralityof oligonucleotides attached to the substrate in the array is locatedbetween two electrodes, the nanoparticles are made of a material whichis a conductor of electricity, and the detectable change is a change inconductivity.
 382. The method of claim 381 wherein the electrodes aremade of gold, and the nanoparticles are made of gold.
 383. The method ofclaim 381 wherein the substrate is contacted with silver stain toproduce the change in conductivity.
 384. A method of detecting a nucleicacid having at least two portions comprising: (a) contacting the nucleicacid with a substrate having oligonucleotides attached thereto, theoligonucleotides having a sequence complementary to a first portion ofthe sequence of said nucleic acid, the contacting taking place underconditions effective to allow hybridization of the oligonucleotides onthe substrate with said nucleic acid; (b) contacting said nucleic acidbound to the substrate with a first type of nanoparticles according toany one of claims 253-263 having one or more types of recognitionoligonucleotides attached thereto, at least one of the types ofrecognition oligonucleotides comprising a sequence complementary to asecond portion of the sequence of said nucleic acid, the contactingtaking place under conditions effective to allow hybridization of therecognition oligonucleotides on the nanoparticles with said nucleicacid; and (c) observing a detectable change.
 385. The method of claim384 further comprising: (d) contacting the first type of nanoparticlesbound to the substrate with a second type of nanoparticles according toany one of claims 253-263 having recognition oligonucleotides attachedthereto, at least one of the types of recognition oligonucleotides onthe second type of nanoparticles comprising a sequence complementary tothe sequence of one of the types of oligonucleotides on the first typeof nanoparticles, the contacting taking place under conditions effectiveto allow hybridization of the oligonucleotides on the first and secondtypes of nanoparticles; and (e) observing the detectable change. 386.The method of claim 385 wherein at least one of the types of recognitionoligonucleotides on the first type of nanoparticles comprises a sequencecomplementary to the sequence of at least one of the types ofoligonucleotides on the second type of nanoparticles and the methodfurther comprises: (f) contacting the second type of nanoparticles boundto the substrate with the first type of nanoparticles, the contactingtaking place under conditions effective to allow hybridization of theoligonucleotides on the first and second types of nanoparticles; and (g)observing the detectable change.
 387. The method of claim 386 whereinstep (d) or steps (d) and (f) are repeated one or more times and thedetectable change is observed.
 388. The method of claim 384 furthercomprising: (d) providing a type of binding oligonucleotides having asequence comprising at least two portions, the first portion beingcomplementary to at least one of the types of oligonucleotides on thefirst type of nanoparticles; (e) contacting the binding oligonucleotideswith the first type of nanoparticles bound to the substrate, thecontacting taking place under conditions effective to allowhybridization of the binding oligonucleotides with the oligonucleotideson the first type of nanoparticles; (f) providing a second type ofnanoparticles according to any one of claims 253-263 having recognitionoligonucleotides attached thereto, at least one of the types ofrecognition oligonucleotides on the second type of nanoparticlescomprising a sequence complementary to the second portion of thesequence of the binding oligonucleotides; (g) contacting the bindingoligonucleotides bound to the substrate with the second type ofnanoparticles, the contacting taking place under conditions effective toallow hybridization of the oligonucleotides on the second type ofnanoparticles with the binding oligonucleotides; and (h) observing thedetectable change.
 389. The method of claim 388 further comprising: (i)contacting the second type of nanoparticles bound to the substrate withthe binding oligonucleotides, the contacting taking place underconditions effective to allow hybridization of the bindingoligonucleotides with the oligonucleotides on the second type ofnanoparticles; (j) contacting the binding oligonucleotides bound to thesubstrate with the first type of nanoparticles, the contacting takingplace under conditions effective to allow hybridization of theoligonucleotides on the first type of nanoparticles with the bindingoligonucleotides; and (k) observing the detectable change.
 390. Themethod of claim 389 wherein steps (e) and (g) or steps (e), (g), (i) and(j) are repeated one or more times, and the detectable change isobserved.
 391. The method of claim 384 wherein the substrate is atransparent substrate or an opaque white substrate.
 392. The method ofclaim 391 wherein the detectable change is the formation of dark areason the substrate.
 393. The method of claim 384 wherein the nanoparticlesare metal nanoparticles or semiconductor nanoparticles.
 394. The methodof claim 393 wherein the nanoparticles are made of gold or silver. 395.The method of claim 384 wherein the substrate has a plurality of typesof oligonucleotides attached to it in an array to allow for thedetection of multiple portions of a single nucleic acid, the detectionof multiple different nucleic acids, or both.
 396. The method of claim384 wherein the substrate is contacted with silver stain to produce thedetectable change.
 397. The method of claim 395 wherein the substrate iscontacted with silver stain to produce the detectable change.
 398. Themethod of claim 384 wherein the detectable change is observed with anoptical scanner
 399. The method of claim 398 wherein the device is aflatbed scanner.
 400. The method of claim 398 wherein the scanner islinked to a computer loaded with software capable of calculatinggreyscale measurements, and the greyscale measurements are calculated,to provide a quantitative measure of the amount of nucleic aciddetected.
 401. The method of claim 384 wherein the oligonucleotidesattached to the substrate are located between two electrodes, thenanoparticles are made of a material which is a conductor ofelectricity, and the detectable change is a change in conductivity. 402.The method of claim 401 wherein the electrodes are made of gold, and thenanoparticles are made of gold.
 403. The method of claim 401 wherein thesubstrate is contacted with silver stain to produce the change inconductivity.
 404. The method of claim 397 wherein each of the pluralityof oligonucleotides attached to the substrate in the array is locatedbetween two electrodes, the nanoparticles are made of a material whichis a conductor of electricity, and the detectable change is a change inconductivity.
 405. The method of claim 404 wherein the electrodes aremade of gold, and the nanoparticles are made of gold.
 406. The method ofclaim 404 wherein the substrate is contacted with silver stain toproduce the change in conductivity.
 407. A method of detecting a nucleicacid having at least two portions comprising: (a) contacting the nucleicacid with a substrate having oligonucleotides attached thereto, theoligonucleotides being located between a pair of electrodes, theoligonucleotides having a sequence complementary to a first portion ofthe sequence of said nucleic acid, the contacting taking place underconditions effective to allow hybridization of the oligonucleotides onthe substrate with said nucleic acid; (b) contacting said nucleic acidbound to the substrate with a first type of nanoparticles, thenanoparticles being made of a material which can conduct electricity,the nanoparticles having one or more types of oligonucleotides attachedthereto, at least one of the types of oligonucleotides having a sequencecomplementary to a second portion of the sequence of said nucleic acid,the contacting taking place under conditions effective to allowhybridization of the oligonucleotides on the nanoparticles with saidnucleic acid; and (c) detecting a change in conductivity.
 408. Themethod of claim 407 wherein the substrate has a plurality of pairs ofelectrodes located on it in an array to allow for the detection ofmultiple portions of a single nucleic acid, the detection of multipledifferent nucleic acids, or both, each of the pairs of electrodes havinga type of oligonucleotides attached to the substrate between them. 409.The method of claim 407 wherein the nanoparticles are made of metal.410. The method of claim 407 wherein the nanoparticles are made of goldor silver.
 411. The method of claim 407 wherein the substrate iscontacted with silver stain to produce the change in conductivity. 412.The method of claim 407 further comprising: (d) contacting the firsttype of nanoparticles bound to the substrate with a second type ofnanoparticles, the nanoparticles being made of a material which canconduct electricity, the nanoparticles having oligonucleotides attachedthereto, at least one of the types of oligonucleotides on the secondtype of nanoparticles comprising a sequence complementary to thesequence of one of the types of oligonucleotides on the first type ofnanoparticles, the contacting taking place under conditions effective toallow hybridization of the oligonucleotides on the first and secondtypes of nanoparticles; and (e) detecting the change in conductivity.413. The method of claim 412 wherein at least one of the types ofoligonucleotides on the first type of nanoparticles has a sequencecomplementary to the sequence of at least one of the types ofoligonucleotides on the second type of nanoparticles and the methodfurther comprises: (f) contacting the second type of nanoparticles boundto the substrate with the first type of nanoparticles, the contactingtaking place under conditions effective to allow hybridization of theoligonucleotides on the first and second types of nanoparticles; and (g)detecting the change in conductivity.
 414. The method of claim 413wherein step (d) or steps (d) and (f) are repeated one or more times andthe change in conductivity is detected. 415 The method of claim 407further comprising: (d) contacting the first type of nanoparticles boundto the substrate with an aggregate probe having oligonucleotidesattached thereto, the nanoparticles of the aggregate probe being made ofa material which can conduct electricity, at least one of the types ofoligonucleotides on the aggregate probe comprising a sequencecomplementary to the sequence of one of the types of oligonucleotides onthe first type of nanoparticles, the contacting taking place underconditions effective to allow hybridization of the oligonucleotides onthe aggregate probe with the oligonucleotides on the first type ofnanoparticles; (e) and detecting the change in conductivity.
 416. Amethod of detecting nucleic acid having at least two portionscomprising: (a) contacting a nucleic acid with a substrate havingoligonucleotides attached thereto, the oligonucleotides being locatedbetween a pair of electrodes, the oligonucleotides having a sequencecomplementary to a first portion of the sequence of said nucleic acid,the contacting taking place under conditions effective to allowhybridization of the oligonucleotides on the substrate with said nucleicacid; (b) contacting said nucleic acid bound to the substrate with anaggregate probe having oligonucleotides attached thereto, at least oneof the types of oligonucleotides on the aggregate probe comprising asequence complementary to the sequence of a second portion of saidnucleic acid, the nanoparticles of the aggregate probe being made of amaterial which can conduct electricity, the contacting taking placeunder conditions effective to allow hybridization of theoligonucleotides on the aggregate probe with the nucleic acid; and (c)detecting a change in conductivity.
 417. A method of detecting a nucleicacid wherein the method is performed on a substrate, the methodcomprising detecting the presence, quantity, or both, of the nucleicacid with an optical scanner.
 418. The method of claim 417 wherein thedevice is a flatbed scanner.
 419. The method of claim 417 wherein thescanner is linked to a computer loaded with software capable ofcalculating greyscale measurements, and the greyscale measurements arecalculated. to provide a quantitative measure of the amount of nucleicacid detected.
 420. The method of claim 417 wherein the scanner islinked to a computer loaded with software capable of providing an imageof the substrate, and a qualitative determination of the presence of thenucleic acid, the quantity of the nucleic acid, or both, is made.
 421. Akit comprising a container holding nanoparticle-oligonucleotideconjugates according to any one of claims 237-242.
 422. A kit comprisinga container holding nanoparticles according to any one of claims243-265.
 423. A kit comprising a substrate having attached thereto atleast one pair of electrodes with oligonucleotides attached to thesubstrate between the electrodes.
 424. The kit of claim 423 wherein thesubstrate has a plurality of pairs of electrodes attached to it in anarray, to allow for the detection of multiple portions of a singlenucleic acid, the detection of multiple different nucleic acids, orboth.
 425. A method of nanofabrication comprising providing at least onetype of linking oligonucleotide having a selected sequence, the sequenceof each type of linking oligonucleotide having at least two portions;providing one or more types of nanoparticle-oligonucleotide conjugatesaccording to any one of claims 237-242, the oligonucleotides attached tothe nanoparticles of each of the types of conjugates having a sequencecomplementary to the sequence of a portion of a linking oligonucleotide;and contacting the linking oligonucleotides and conjugates underconditions effective to allow hybridization of the oligonucleotidesattached to the nanoparticles of the conjugates to the linkingoligonucleotides so that a desired nanomaterial or nanostructure isformed wherein the nanoparticles of the conjugates are held together byoligonucleotide connectors.
 426. A method of nanofabrication comprisingproviding at least one type of linking oligonucleotide having a selectedsequence, the sequence of each type of linking oligonucleotide having atleast two portions; providing one or more types of nanoparticlesaccording to any one of claims 243-265, the recognition oligonucleotideson each of the types of nanoparticles comprising a sequencecomplementary to the sequence of a portion of a linking oligonucleotide;and contacting the linking oligonucleotides and nanoparticles underconditions effective to allow hybridization of the oligonucleotides onthe nanoparticles to the linking oligonucleotides so that a desirednanomaterial or nanostructure is formed wherein the nanoparticles areheld together by oligonucleotide connectors.
 427. A method ofnanofabrication comprising: providing at least two types ofnanoparticle-oligonucleotide conjugates according to any one of claims237-242, the oligonucleotides attached to the nanoparticles of the firsttype of conjugates having a sequence complementary to that of theoligonucleotides attached to the nanoparticles of the second type ofconjugates; the oligonucleotides attached to the nanoparticles of thesecond type of conjugates having a sequence complementary to that of theoligonucleotides attached to the nanoparticles of the first type ofconjugates; and contacting the first and second types of conjugatesunder conditions effective to allow hybridization of theoligonucleotides on the nanoparticles of the conjugates to each other sothat a desired nanomaterial or nanostructure is formed.
 428. A method ofnanofabrication comprising: providing at least two types ofnanoparticles according to any one of claims 243-265, the recognitionoligonucleotides on the first type of nanoparticles comprising asequence complementary to that of the oligonucleotides on the second ofthe nanoparticles; the recognition oligonucleotides on the second typeof nanoparticles comprising a sequence complementary to that of theoligonucleotides on the first type of nanoparticles; and contacting thefirst and second types of nanoparticles under conditions effective toallow hybridization of the oligonucleotides on the nanoparticles to eachother so that a desired nanomaterial or nanostructure is formed. 429.Nanomaterials or nanostructures composed of nanoparticle-oligonucleotideconjugates according to any one of claims 237-242, the nanoparticlesbeing held together by oligonucleotide connectors.
 430. Nanomaterials ornanostructures composed of nanoparticles according to any one of claims243-265, the nanoparticles being held together by oligonucleotideconnectors.
 431. A method of separating a selected nucleic acid havingat least two portions from other nucleic acids, the method comprising:providing two or more types of nanoparticle-oligonucleotide conjugatesaccording to any one of claims 237-242, the oligonucleotides attached tothe nanoparticles of each of the types of conjugates having a sequencecomplementary to the sequence of one of the portions of the selectednucleic acid; and contacting the nucleic acids and conjugates underconditions effective to allow hybridization of the oligonucleotides onthe nanoparticles of the conjugates with the selected nucleic acid sothat the conjugates hybridized to the selected nucleic acid aggregateand precipitate.
 432. A method of separating a selected nucleic acidhaving at least two portions from other nucleic acids, the methodcomprising: providing two or more types of nanoparticles according toany one of claims 243-265, the oligonucleotides on each of the types ofnanoparticles having a sequence complementary to the sequence of one ofthe portions of the selected nucleic acid; and contacting the nucleicacids and nanoparticles under conditions effective to allowhybridization of the oligonucleotides on the nanoparticles with theselected nucleic acid so that the nanoparticles hybridized to theselected nucleic acid aggregate and precipitate.